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Read, PJ, Oliver, JL, de Ste Croix, MBA, Myer, GD, and Lloyd, RS. Reliability of the tuck jump injury risk screening assessment in elite male youth soccer players. J Strength Cond Res 30(6): 1510-1516, 2016-Altered neuromuscular control has been suggested as a mechanism for injury in soccer players. Ligamentous injuries most often occur during dynamic movements, such as decelerations from jump-landing maneuvers where high-risk movement patterns are present. The assessment of kinematic variables during jump-landing tasks as part of a preparticipation screen is useful in the identification of injury risk. An example of a field-based screening tool is the repeated tuck jump assessment. The purpose of this study was to analyze the within-subject variation of the tuck jump screening assessment in elite male youth soccer players. Twenty-five pre-peak height velocity (PHV) and 25 post-PHV elite male youth soccer players from the academy of a professional English soccer club completed the assessment. A test-retest design was used to explore the within-subject intersession reliability. Technique was graded retrospectively against the 10-point criteria set out in the screening protocol using two-dimensional video cameras. The typical error range reported for tuck jump total score (0.90-1.01 in pre-PHV and post-PHV players respectively) was considered acceptable. When each criteria was analyzed individually, kappa coefficient determined that knee valgus was the only criterion to reach substantial agreement across the two test sessions for both groups. The results of this study suggest that although tuck jump total score may be reliably assessed in elite male youth soccer players, caution should be applied in solely interpreting the composite score due to the high within-subject variation in a number of the individual criteria. Knee valgus may be reliably used to screen elite youth male soccer players for this plyometric technique error and for test-retest comparison.

There has recently been a growing interest in long-term athletic development for youth. Because of their unique physical, psychological, and social differences, children and adolescents should engage in appropriately prescribed exercise programs that promote physical development to prevent injury and enhance fitness behaviors that can be retained later in life. Irrespective of whether a child is involved in organized sport or engages in recreational physical activity, there remains a need to adopt a structured, logical, and evidence-based approach to the long-term development of athleticism. This is of particular importance considering the alarmingly high number of youth who fail to meet global physical activity recommendations and consequently present with negative health profiles. However, appropriate exercise prescription is also crucial for those young athletes who are physically underprepared and at risk of overuse injury because of high volumes of competition and an absence of preparatory conditioning. Whether the child accumulates insufficient or excessive amounts of exercise, or falls somewhere between these opposing ends of the spectrum, it is generally accepted that the young bodies of modern day youth are often ill-prepared to tolerate the rigors of sports or physical activity. All youth should engage in regular physical activity and thus should be viewed as "athletes" and afforded the opportunity to enhance athleticism in an individualized, holistic, and child-centered manner. Because of emerging interest in long-term athletic development, an authorship team was tasked on behalf of the National Strength and Conditioning Association (NSCA) to critically synthesize existing literature and current practices within the field and to compose a relevant position statement. This document was subsequently reviewed and formally ratified by the NSCA Board of Directors. A list of 10 pillars of successful long-term athletic development are presented, which summarize the key recommendations detailed within the position statement. With these pillars in place, it is believed that the NSCA can (a) help foster a more unified and holistic approach to long-term athletic development, (b) promote the benefits of a lifetime of healthy physical activity, and, ((c) prevent and/or minimize injuries from sports participation for all boys and girls.)

The purpose of this study was to compare the effectiveness of 6-week training interventions using different modes of resistance (traditional strength, plyometric, and combined training) on sprinting and jumping performances in boys before and after peak height velocity (PHV). Eighty school-aged boys were categorized into 2 maturity groups (pre- or post-PHV) and then randomly assigned to (a) plyometric training, (b) traditional strength training, (c) combined training, or (d) a control group. Experimental groups participated in twice-weekly training programs for 6 weeks. Acceleration, maximal running velocity, squat jump height, and reactive strength index data were collected pre- and postintervention. All training groups made significant gains in measures of sprinting and jumping irrespective of the mode of resistance training and maturity. Plyometric training elicited the greatest gains across all performance variables in pre-PHV children, whereas combined training was the most effective in eliciting change in all performance variables for the post-PHV cohort. Statistical analysis indicated that plyometric training produced greater changes in squat jump and acceleration performances in the pre-PHV group compared with the post-PHV cohort. All other training responses between pre- and post-PHV cohorts were not significant and not clinically meaningful. The study indicates that plyometric training might be more effective in eliciting short-term gains in jumping and sprinting in boys who are pre-PHV, whereas those who are post-PHV may benefit from the additive stimulus of combined training.

The motto of the Olympic Games is Citius, Altius, Fortius which is Latin for 'Faster, Higher, Stronger'. It is a clarion call to all competitors, including the youngest, to engage in training strategies that prepare athletes to be the best in the world. Existing research indicates that various forms of resistance training can elicit performance improvements in young athletes. Stronger young athletes will be better prepared to learn complex movements, master sport tactics, and sustain the demands of training and competition. An integrative training programme grounded in resistance training and motor skill development can optimise a young athlete's potential to maximise their athletic and sporting performance, while reducing the risk of a sports-related injury. Resistance training may be especially important for modern-day young athletes who are more likely to specialise in one sport at an early age at the expense of enhancing general physical fitness and learning diversified sport skills. Structured interventions that include qualified instruction; targeted movement practice; and strength and conditioning activities that are developmentally appropriate, progressive and technique driven are needed to attain a level of athleticism that is consistent with the Olympic motto., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

The DNA base excision repair (BER) pathway, which utilizes DNA glycosylases to initiate repair of specific DNA lesions, is the major pathway for the repair of DNA damage induced by oxidation, alkylation, and deamination. Early results from clinical trials suggest that inhibiting certain enzymes in the BER pathway can be a useful anticancer strategy when combined with certain DNA-damaging agents or tumor-specific genetic deficiencies. Despite this general validation of BER enzymes as drug targets, there are many enzymes that function in the BER pathway that have few, if any, specific inhibitors. There is a growing body of evidence that suggests inhibition of 8-oxoguanine DNA glycosylase-1 (OGG1) could be useful as a monotherapy or in combination therapy to treat certain types of cancer. To identify inhibitors of OGG1, a fluorescence-based screen was developed to analyze OGG1 activity in a high-throughput manner. From a primary screen of ∼50,000 molecules, 13 inhibitors were identified, 12 of which were hydrazides or acyl hydrazones. Five inhibitors with an IC50 value of less than 1 μM were chosen for further experimentation and verified using two additional biochemical assays. None of the five OGG1 inhibitors reduced DNA binding of OGG1 to a 7,8-dihydro-8-oxoguanine (8-oxo-Gua)-containing substrate, but all five inhibited Schiff base formation during OGG1-mediated catalysis. All of these inhibitors displayed a >100-fold selectivity for OGG1 relative to several other DNA glycosylases involved in repair of oxidatively damaged bases. These inhibitors represent the most potent and selective OGG1 inhibitors identified to date.

Fatigue is known to influence dynamic knee joint stability from a neuromuscular perspective, and electromechanical delay (EMD) plays an important role as the feedback activation mechanism that stabilizes the joint. The aim of this study was to investigate the influence of soccer-specific fatigue on EMD in U13-, U15-, and U17-year-old female soccer players. Thirty-six youth soccer players performed eccentric actions of the hamstrings in a prone position at 60, 120, and 180°/s before and after a soccer-specific fatigue trial. Surface electromyography was used to determine EMD from the semitendinosus, biceps femoris and gastrocnemius. A time × age × muscle × velocity repeated measures analysis of variance was used to explore the influence of fatigue on EMD. A significant main effect for time (P = 0.001) indicated that EMD was significantly longer post- compared with pre-fatigue (58.4% increase). A significant time × group interaction effect (P = 0.046) indicated EMD was significantly longer in the U13 age group compared with the U15 (P = 0.011) and U17 (P = 0.021) groups and greater post-fatigue. Soccer-specific fatigue compromised neuromuscular feedback mechanisms and the age-related effects may represent a more compliant muscle-tendon system in younger compared with older girls, increasing risk of injury., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The purpose of this study was to examine the reliability of the spatiotemporal determinants of maximal sprinting speed in boys over single and multiple steps. Fifty-four adolescent boys (age = 14.1 ± 0.7 years [range = 12.9-15.7 years]; height = 1.63 ± 0.09 m; body mass = 55.3 ± 13.3 kg; -0.31 ± 0.90 age from Peak Height Velocity (PHV) in years; mean ± s) volunteered to complete a 30 m sprint test on 3 occasions over a 2-week period. Speed, step length, step frequency, contact time, and flight time were assessed via an optical measurement system. Speed and step characteristics were obtained from the single-fastest step and average of the 2 and 4 fastest consecutive steps. Pairwise comparison of consecutive trials revealed the coefficient of variation (CV) for speed was greater in 4-step (CV = 7.3 & 7.5%) compared with 2-step (CV = 4.2 & 4.1%) and 1-step (CV = 4.8 & 4.6%) analysis. The CV of step length, step frequency and contact time ranged from 4.8 to 7.5% for 1-step, 3.8-5.0% for 2-step and 4.2-7.5% for 4-step analyses across all trials. An acceptable degree of reliability was achieved for the spatiotemporal and performance variables assessed in this study. Two-step analysis demonstrated the highest degree of reliability for the key spatiotemporal variables, and therefore may be the most suitable approach to monitor the spatiotemporal characteristics of maximal sprint speed in boys.

The concept of developing talent and athleticism in youth is the goal of many coaches and sports systems. Consequently, an increasing number of sporting organizations have adopted long-term athletic development models in an attempt to provide a structured approach to the training of youth. It is clear that maximizing sporting talent is an important goal of long-term athletic development models. However, ensuring that youth of all ages and abilities are provided with a strategic plan for the development of their health and physical fitness is also important to maximize physical activity participation rates, reduce the risk of sport- and activity-related injury, and to ensure long-term health and well-being. Critical reviews of independent models of long-term athletic development are already present within the literature; however, to the best of our knowledge, a comprehensive examination and review of the most prominent models does not exist. Additionally, considerations of modern day issues that may impact on the success of any long-term athletic development model are lacking, as are proposed solutions to address such issues. Therefore, within this 2-part commentary, Part 1 provides a critical review of existing models of practice for long-term athletic development and introduces a composite youth development model that includes the integration of talent, psychosocial and physical development across maturation. Part 2 identifies limiting factors that may restrict the success of such models and offers potential solutions.

The first installment of this two-part commentary reviewed existing models of long-term athletic development. However, irrespective of the model that is adopted by practitioners, existing structures within competitive youth sports in addition to the prevalence of physical inactivity in a growing number of modern-day youth may serve as potential barriers to the success of any developmental pathway. The second part of this commentary will initially highlight common issues that are likely to impede the success of long-term athletic development programs and then propose solutions that will address the negative impact of such issues.

The back squat is a well-researched and widely used exercise to enhance fundamental movement competency that creates a foundation for optimal mechanical strategies during a broad range of activities. The primary commentary introduced the Back Squat Assessment (BSA): a criterion based assessment of the back squat that delineates 30 potentially observable functional deficits. This follow-up commentary provides a targeted system of training cues and exercises to supplement the BSA to guide corrective intervention. We propose a criterion driven approach to corrective exercise that can support practitioners in their goal to help individuals achieve movement competency in the back squat.

The purpose of this study was to examine the natural development of the mechanical features of sprint performance in relation to maturation within a large cohort of boys. Three hundred and thirty-six boys (11-15 years) were analyzed for sprint performance and maturation. Maximal speed, stride length (SL), stride frequency (SF), flight time (FT) and contact time (CT) were assessed during a 30m sprint. Five maturation groups (G1-5) were established based on age from peak height velocity (PHV) where G1=>2.5years pre-PHV, G2 = 2.49-1.5years pre-PHV, G3 = 1.49-0.5years pre-PHV, G4 = 0.49years pre- to 0.5years post-PHV and G5 = 0.51-1.5years post-PHV. There was no difference in maximal speed between G1, G2 and G3 but those in G4 and G5 were significantly faster (p < .05) than G1-3. Significant increases (p < .05) in SL were observed between groups with advancing maturation, except G4 and G5 (p > .05). SF decreased while CT increased (both p < .05) between G1, G2 and G3, but no further significant changes (p > .05) were observed for either variable between G3, G4 and G5. While G1-3 increased their SL, concomitant decreases in SF and increases in CT prevented them from improving maximal speed. Maximal sprint speed appears to develop around and post-PHV as SF and CT begin to stabilize, with increases in maximal sprint speed in maturing boys being underpinned by increasing SL.

The purpose of this study was to examine relationships between functional movement screen scores, maturation and physical performance in young soccer players. Thirty males (11-16 years) were assessed for maturation, functional movement screen scores and a range of physical performance tests (squat jump, reactive strength index protocol and reactive agility cut). Older players significantly outperformed younger participants in all tests (P < 0.05; effect sizes = 1.25-3.40). Deep overhead squat, in-line lunge, active straight leg raise and rotary stability test were significantly correlated to all performance tests. In-line lunge performance explained the greatest variance in reactive strength index (adjusted R(2) = 47%) and reactive agility cut (adjusted R(2) = 38%) performance, whilst maturation was the strongest predictor of squat jump performance (adjusted R(2) = 46%). This study demonstrated that variation of physical performance in youth soccer players could be explained by a combination of both functional movement screen scores and maturation.

The purpose of this study was to examine both short- and long-term neuromuscular and perceptual fatigue in youth rugby players during a seven-week in-season mesocycle. Eleven male youth rugby players (age 16.9 ± 0.8 years) were assessed for countermovement jump (CMJ), reactive strength index (RSI) and leg stiffness to monitor neuromuscular performance, together with a well-being questionnaire to monitor perceptual fatigue. Players trained and competed throughout a seven-week block with test variables measured at baseline and 24 h pre- and post-matches played in weeks 1, 4 and 7. Players trained on average 9.7 ± 1.1 h per week and competed in 10.5 ± 1.9 games over the seven-week block. Pre- to post-match reductions were significant across all games for CMJ, RSI and well-being (all P < 0.05), ranging from likely to almost certain negative reductions. Well-being and RSI demonstrated non-significant (P < 0.05) unclear or inconsistent changes when comparing pre-match scores to baseline. Significant reductions to baseline were observed pre-match for CMJ (weeks 4 and 7) and stiffness (week 7), representing very likely to almost certain negative long-term decrements. CMJ, RSI and well-being were all sensitive to detecting post-match fatigue. Importantly, CMJ and stiffness were sensitive to detecting accumulated fatigue over a seven-week period, whereas RSI and well-being were not. Consequently, either a CMJ or leg stiffness should be monitored to detect long-term, accumulated fatigue in academy rugby players.

Fundamental movement competency is essential for participation in physical activity and for mitigating the risk of injury, which are both key elements of health throughout life. The squat movement pattern is arguably one of the most primal and critical fundamental movements necessary to improve sport performance, to reduce injury risk and to support lifelong physical activity. Based on current evidence, this first (1 of 2) report deconstructs the technical performance of the back squat as a foundation training exercise and presents a novel dynamic screening tool that incorporates identification techniques for functional deficits that limit squat performance and injury resilience. The follow-up report will outline targeted corrective methodology for each of the functional deficits presented in the assessment tool.

Purpose: To examine changes to neuromuscular control of leg stiffness following 42 min of soccer-specific exercise., Methods: Ten youth soccer players, aged 15.8 ± 0.4 years, stature 1.73 ± 0.06 m and mass 59.8 ± 9.7 kg, hopped on a force plate at a self-selected frequency before and after simulated soccer exercise performed on a non-motorised treadmill. During hopping, muscle activity was measured using surface electromyography from four lower limb muscles and analysed to determine feedforward- and feedback-mediated activity, as well as co-contraction., Results: There was a small, non-significant change in stiffness following exercise (26.6 ± 10.6 vs. 24.0 ± 7.0 kN m(-1), p > 0.05, ES = 0.25), with half the group increasing and half decreasing their stiffness. Changes in stiffness were significantly related to changes in centre of mass (CoM) displacement (r = 0.90, p < 0.01, extremely large correlation) but not changes in peak ground reaction force (r = 0.58, p > 0.05, large correlation). A number of significant relationships were observed between changes in stiffness and CoM displacement with changes in feedforward, feedback and eccentric muscle activity of the soleus and vastus lateralis muscles following exercise (r = 0.64-0.98, p < 0.05, large-extremely large correlations), but not with changes in co-contraction (r = 0.11-0.55, p > 0.05, small-large correlations)., Conclusions: Following soccer-specific exercise individual changes in feedforward- and reflex-mediated activity of the soleus and vastus lateralis, and not co-contraction around the knee and ankle, modulate changes in CoM displacement and leg stiffness.

Site-specifically modified DNAs are routinely used in the study of DNA damage-induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a pre-determined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively labeled probes, and verification of mutations by Sanger sequencing. In the search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site-specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N(6) -(deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG)-containing vectors in primate cells, with the lesion being positioned in the 5'-GCNGG-3' sequence context. Pyrosequencing detected ∼8% G to T transversions and ∼3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure (Earley LF et al. [2013]: Chem Res Toxicol 26:1108-1114). However, ∼3.5% G to C transversions and ∼2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ∼1-2% for A and T and ∼5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy-dG-containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage-induced mutagenesis as an effective complementary experimental approach to current protocols., (© 2014 Wiley Periodicals, Inc.)

Aflatoxin B1 (AFB1) is a known carcinogen associated with early-onset hepatocellular carcinoma (HCC) and is thought to contribute to over half a million new HCCs per year. Although some of the fundamental risk factors are established, the molecular basis of AFB1-induced mutagenesis in primate cells has not been rigorously investigated. To gain insights into genome instability that is produced as a result of replicating DNAs containing AFB1 adducts, site-specific mutagenesis assays were used to establish the mutagenic potential of the persistent ring-opened AFB1 adduct, AFB1-formamidopyrimidine (AFB1-FAPY). This lesion was highly mutagenic, yielding replication error frequencies of 97%, with the predominant base substitution being a G to T transversion. This transversion is consistent with previous mutational data derived from aflatoxin-associated HCCs. In vitro translesion synthesis assays demonstrated that polymerase (pol) ζ was the most likely candidate polymerase that is responsible for the G to T mutations induced by this adduct., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hepatocellular carcinomas (HCCs) are the third leading cause of cancer deaths worldwide. The highest rates of early onset HCCs occur in geographical regions with high aflatoxin B1 (AFB1) exposure, concomitant with hepatitis B infection. Although the carcinogenic basis of AFB1 has been ascribed to its mutagenic effects, the mutagenic property of the primary AFB1-DNA adduct, AFB1-N7-Gua, in mammalian cells has not been studied extensively. Taking advantage of the ability to create vectors containing a site-specific DNA adduct, the mutagenic potential was determined in primate cells. This adduct was highly mutagenic following replication in COS-7 cells, with a mutation frequency of 45%. The spectrum of mutations was predominantly G to T base substitutions, a result that is consistent with previous mutation data derived from aflatoxin-associated HCCs. To assess which DNA polymerases (pol) might contribute to the mutational outcome, in vitro replication studies were performed. Unexpectedly, replicative pol δ and the error-prone translesion synthesis pol ζ were able to accurately bypass AFB1-N7-Gua. In contrast, replication bypass using pol κ was shown to occur with low fidelity and could account for the commonly detected G to T transversions., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Biological maturation is associated with significant change to a number of physiological and structural processes throughout childhood and, in particular, adolescence. Mismatched rapid growth in the long bones relative to muscular lengthening may disrupt structure, neuromuscular function, and physical performance. Practitioners who work with school-age youth should be aware of the age-related changes that typically take place during a child's development to ensure that their strength and conditioning programming is as safe and effective as possible for enhancing performance and reducing injury risk. Although there are several methods available to assess biological maturation, practitioners who work with youth can benefit from assessment methods that are available and feasible, and that provide utility in the quantification of the degree and stages of biological maturation that affect motor performance in children and adolescents. This article synthesizes the relevant assessment methods and provides a rationale for understanding usable biological maturation assessment tools that can aid in the development of training program design for youth.

The current manuscript has been adapted from the official position statement of the UK Strength and Conditioning Association on youth resistance training. It has subsequently been reviewed and endorsed by leading professional organisations within the fields of sports medicine, exercise science and paediatrics. The authorship team for this article was selected from the fields of paediatric exercise science, paediatric medicine, physical education, strength and conditioning and sports medicine.

Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.

Since the publication of the seminal review on youth resistance training by Kraemer and colleagues in 1989, a compelling body of evidence has found that resistance training can be a safe, effective, and worthwhile method of conditioning for children and adolescents. New perspectives for promoting resistance exercise as part of a long-term approach to youth physical development highlight the importance of integrating resistance training into youth fitness programs. Youth who do not enhance their muscular strength and motor skill proficiency early in life may not develop the prerequisite skills and abilities that would allow them to participate in a variety of activities and sports with confidence and vigor later in life. The identification of asymptomatic children with muscular weaknesses or imbalances may facilitate the development of a management plan which should rectify movement limitations and educate children and their families about the importance of daily physical activity.

DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N(6) -BPDE dA adducts at the second position in the N-ras codon 61(CAA) (61(2) ) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C10 S-BPDE and C10 R-BPDE lesions linked through the N(6) position of adenine in the sequence context N-ras codon 61, position 3 (C10 S-BPDE and C10 R-BPDE at 61(3) ) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C10 S-BPDE and C10 R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 61(3) resulted in truncated products, with the C10 S-BPDE adducts being more blocking than C10 R-BPDE lesions, and termination of synthesis was more pronounced at position 61(3) than at 61(2) for each of the lesions., (Copyright © 2013 Wiley Periodicals, Inc.)

Increased golf club head speed (CHS) has been shown to result in greater driving distances and is also correlated with golf handicap. The purpose of this study was to investigate the relationships between field-based measures of strength and power and golf CHS with a secondary aim to determine the reliability of the selected tests. A correlation design was used to assess the following variables: anthropometrics, squat jump (SJ) height and squat jump peak power (SJPP), unilateral countermovement jump (CMJ) heights (right leg countermovement jump and left leg countermovement jump [LLCMJ]), bilateral CMJ heights, countermovement jump peak power (CMJPP), and medicine ball seated throw (MBST) and medicine ball rotational throw (MBRT). Fouty-eight male subjects participated in the study (age: 20.1 ± 3.2 years, height: 1.76 ± 0.07 m, mass: 72.8 ± 7.8 kg, handicap: 5.8 ± 2.2). Moderate significant correlations were reported between CHS and MBRT (r = 0.67; p < 0.01), MBST (r = 0.63; p < 0.01), CMJPP (r = 0.54; p < 0.01), and SJPP (r = 0.53; p < 0.01). Weak significant correlations (r = 0.3-0.5) were identified between CHS and the other remaining variables excluding LLCMJ. Stepwise multiple regression analysis identified that the MBST and SJ were the greatest predictors of CHS, explaining 49% of the variance. Additionally the intraclass correlation coefficients reported for tests of CHS and all performance variables were deemed acceptable (r = 0.7-0.9). The results of this study suggest that the strength and conditioning coach can accurately assess and monitor the physical abilities of golf athletes using the proposed battery of field tests. Additionally, movements that are more concentrically dominant in nature may display stronger relationships with CHS due to MBST and SJ displaying the highest explained variance after a stepwise linear regression.

To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT), this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6)-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2'-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one (γ-OH-PdG). When placed opposite deoxycytosine (dC) in DNA, γ-OH-PdG undergoes ring-opening to the N(2)-(3-oxopropyl)-dG. Ring-opening of the adduct has been hypothesized to facilitate nonmutagenic bypass, particularly by DNA polymerases of the Y family. This study examined the bypass of γ-OH-PdG by Sulfolobus solfataricus Dpo4, the prototypic Y-family DNA polymerase, using templates that contained the adduct in either the 5'-CXG-3' or the 5'-TXG-3' sequence context. Although γ-OH-PdG partially blocked Dpo4-catalyzed DNA synthesis, full primer extension was observed, and the majority of bypass products were error-free. Conversion of the adduct into an irreversibly ring-opened derivative prior to reaction facilitated bypass and further improved the fidelity. Structures of ternary Dpo4·DNA·dNTP complexes were determined with primers that either were positioned immediately upstream of the lesion (preinsertion complexes) or had a 3'-terminal dC opposite the lesion (postinsertion complexes); the incoming nucleotides, either dGTP or dATP, were complementary to the template 5'-neighbor nucleotide. In both postinsertion complexes, the adduct existed as ring-opened species, and the resulting base-pair featured Watson-Crick hydrogen bonding. The incoming nucleotide paired with the 5'-neighbor template, while the primer 3'-hydroxyl was positioned to facilitate extension. In contrast, γ-OH-PdG was in the ring-closed form in both preinsertion complexes, and the overall structure did not favor catalysis. These data provide insights into γ-OH-PdG chemistry during replication bypass by the Dpo4 DNA polymerase and may explain why γ-OH-PdG-induced mutations due to primer-template misalignment are uncommon.

DNA exposures to electrophilic methylating agents that are commonly used during chemotherapeutic treatments cause diverse chemical modifications of nucleobases, with reaction at N7-dG being the most abundant. Although this base modification frequently results in destabilization of the glycosyl bond and spontaneous depurination, the adduct can react with hydroxide ion to yield a stable, ring-opened MeFapy-dG, and this lesion has been reported to persist in animal tissues. Results from prior in vitro replication bypass investigations of the MeFapy-dG adduct had revealed complex spectra of replication errors that differed depending on the identity of DNA polymerase and the local sequence context. In this study, a series of nine site-specifically modified MeFapy-dG-containing oligodeoxynucleotides were engineered into a shuttle vector and subjected to replication in primate cells. In all nine sequence contexts examined, MeFapy-dG was shown to be associated with a strong mutator phenotype, predominantly causing base substitutions, with G to T transversions being most common. Single and dinucleotide deletions were also found in a subset of the sequence contexts. Interestingly, single-nucleotide deletions occurred at not only the adducted site, but also one nucleotide downstream of the adduct. Standard models for primer-template misalignment could account for some but not all mutations observed. These data demonstrate that in addition to mutagenesis predicted from replication of DNAs containing O(6)-Me-dG and O(4)-Me-dT, the MeFapy-dG adduct likely contributes to mutagenic events following chemotherapeutic treatments.

Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N(6)-propano-2'-deoxyadenosine (γ-HOPdA). The bulky γ-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized translesion DNA synthesis polymerases to tolerate these lesions. Thus, it is important to understand how pol γ, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of pol γ to bypass the minor groove γ-HOPdG and major groove γ-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of pol γ-catalyzed bypass of γ-HOPdG was low, and surprisingly, pol γ preferred to incorporate purine nucleotides opposite the adduct. Pol γ also exhibited ∼2-fold lower rates of excision of the misincorporated purine nucleotides opposite γ-HOPdG compared with the corresponding nucleotides opposite dG. Extension of primers from the termini opposite γ-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, pol γ preferentially incorporated dT opposite the γ-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that γ-HOPdA is probably nonmutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but, in the case of the minor groove γ-HOPdG adduct, at the cost of unprecedented high mutation rates.

Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC(50) values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis-Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.

Although the primary function of DNA polymerase (pol) β is associated with gap-filling DNA synthesis as part of the DNA base excision repair pathway, translesion synthesis activity has also been described. To further understand the potential role of pol β-catalyzed translesion DNA synthesis (TLS) and the structure-function relationships of specific residues in pol β, wild-type and selected mutants of pol β were used in TLS assays with DNA substrates containing bulky polycyclic aromatic hydrocarbon-adducted oligonucleotides. Stereospecific (+) and (-)-anti-trans-(C(10)S and C(10)R) benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (BPDE) adducts were covalently attached to both the N(6)-adenine and N(2)-guanine in the major and minor grooves, respectively. For all substrates tested, the presence of the BPDE adducts greatly decreased the efficiency of nucleotide incorporation opposite the lesion, and the stereochemistry of the adducts also further modulated the efficiency of the insertion step, such that lesions which were oriented in the 3' direction relative to the approaching polymerase were considerably more blocking than those oriented in the 5' direction. In the absence of a downstream DNA strand, the extension step beyond the adduct was extremely inefficient, relative to a dinucleotide gap-filling reaction, such that in the presence of the downstream DNA, dinucleotide incorporation was strongly favored. In general, analyses of the TLS activities of four pol β mutants revealed similar overall properties, but wild-type pol β exhibited more than 50-fold greater extension and bypass of the C(10)S-dA adducts as compared to a low fidelity mutant R283K expected to interact with the templating base. Replication bypass investigations were further extended to include analyses of HIV-1 reverse transcriptase, and these studies revealed patterns of inhibition very similar to that observed for pol β.

Although previous pediatric research has reported performance improvements in muscular power, agility, speed, and rate-of-force development after exposure to plyometric training, the effects on reactive strength index (RSI) and leg stiffness remain unclear. One hundred and twenty-nine boys from 3 different age groups (9, 12, and 15 years) participated and were divided into either an experimental (EXP) or control (CON) group within their respective age groups. The EXP groups followed a twice-weekly, 4-week plyometric training program, whereas the CON groups participated in their normal physical education lessons. Preintervention and postintervention measures were collected for RSI (during maximal hopping) and leg stiffness (during submaximal hopping). Both 12- and 15-year-old EXP groups made significant improvements in both absolute and relative leg stiffness (p < 0.05). The 9-year-old EXP group and CON groups for all ages did not make significant changes in leg stiffness. The 12-year-old EXP cohort also made significant improvements in RSI (p < 0.05). Both 15- and 9-year-old EXP cohorts, and CON groups for all ages, failed to show any significant improvements in RSI. The study concludes that improvements in RSI and leg stiffness after a 4-week plyometric training program are age dependent during childhood.

N(6)-(2-Deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) has been identified as a stable DNA adduct that arises from the reaction of DNA with a variety of methylating agents. Since this lesion persists in DNA and may contribute to the overall mutagenesis from electrophilic methylating agents, the MeFapy-dGuo lesion was incorporated into oligonucleotides, and its replication bypass was examined in vitro with a panel of eukaryotic high fidelity (hPols α, β, and δ/PCNA) and translesion (hPols η, κ, ι, Rev1, ν, and yPol ζ) polymerases to address its miscoding potential. The MeFapy-dGuo was found to be a strong block to the high fidelity polymerases at either the insertion or the extension step. Efficient translesion synthesis was observed for hPols η and κ, and the combined activities of hRev1 and yPol ζ. The nucleotide sequences of the extension products were determined by mass spectrometry. The error-free extension product was the most abundant product observed for each polymerase. Misreplication products, which included misinsertion of Thy, Gua, and Ade opposite the MeFapy-dGuo lesion, as well as an interesting one-nucleotide deletion product, were observed when hPols η and κ were employed; these events accounted for 8-29% of the total extension products observed. The distribution and abundance of the misreplication products were dependent on the polymerases and local sequence context of the lesion. Collectively, these data suggest that although MeFapy-dGuo adducts represent a relatively minor proportion of the total alkylated lesions, their miscoding potentials could significantly contribute to genomic instability.

This review will present a current understanding of mechanisms for the initiation of base excision repair (BER) of oxidatively-induced DNA damage and the biological consequences of deficiencies in these enzymes in mouse model systems and human populations.

DNA-interstrand cross-links (ICLs) can be repaired by biochemical pathways requiring DNA polymerases that are capable of translesion DNA synthesis (TLS). The anticipated function of TLS polymerases in these pathways is to insert nucleotides opposite and beyond the linkage site. The outcome of these reactions can be either error-free or mutagenic. TLS-dependent repair of ICLs formed between the exocyclic nitrogens of deoxyguanosines (N(2)-dG) can result in low-frequency base substitutions, predominantly G to T transversions. Previously, we demonstrated in vitro that error-free bypass of a model acrolein-mediated N(2)-dG ICL can be accomplished by human polymerase (pol) κ, while Rev1 can contribute to this bypass by inserting dC opposite the cross-linked dG. The current study characterized two additional human DNA polymerases, pol η and pol ι, with respect to their potential contributions to either error-free or mutagenic bypass of these lesions. In the presence of individual dNTPs, pol η could insert dA, dG, and dT opposite the cross-linked dG, but incorporation of dC was not apparent. Further primer extension was observed only from the dC and dG 3' termini, and the amounts of products were low relative to the matched undamaged substrate. Analyses of bypass products beyond the adducted site revealed that dG was present opposite the cross-linked dG in the majority of extended primers, and short deletions were frequently detected. When pol ι was tested for its ability to replicate past this ICL, the correct dC was preferentially incorporated, but no further extension was observed. Under the steady-state conditions, the efficiency of dC incorporation was reduced ~500-fold relative to the undamaged dG. Thus, in addition to pol κ-catalyzed error-free bypass of N(2)-dG ICLs, an alternative, albeit low-efficiency, mechanism may exist. In this pathway, either Rev1 or pol ι could insert dC opposite the lesion, while pol η could perform the subsequent extension., (© 2012 American Chemical Society)

The aim of the current study was to investigate potential age-related differences in neural regulation strategies during maximal and sub-maximal hopping. Thirty-two boys from three different age groups (9-, 12- and 15-years), completed trials of both maximal and sub maximal hopping, and based on contact and flight times, measures of reactive strength index (RSI=jump height/contact time) and leg stiffness (peak ground reaction force/peak displacement of centre of mass) were collected respectively. During all trials, surface electromyograms (EMG) were recorded from four different muscle sites of the dominant lower limb, during 100ms pre-ground contact, and then four subsequent stretch reflex phases: background muscle activity (0-30ms), short-latency stretch reflex (31-60ms), intermediate15 latency stretch reflex 61-90ms and long-latency stretch reflex (91-120ms). Reactive strength index and leg stiffness were measured during the hopping trials. During maximal hopping, both 12- and 15-year olds produced significantly greater RSI (P<0.02) than 9-year olds, with 15-year olds utilising significantly greater soleus muscle activity during the 100ms prior to ground contact than the younger age groups (P<0.01). During sub-maximal hopping, 15-year olds produced significantly greater absolute leg stiffness than both 12- and 9-year olds (P<0.01), with 9-year olds producing significantly less soleus muscle activity during the 31-60ms time phase. For all age groups, sub-maximal hopping was associated with significantly greater background muscle activity and short-latency stretch reflex activity in the soleus and vastus lateralis, when compared to maximal hopping (P<0.001). Results suggest that as children mature, they become more reliant on supra-spinal feed forward input and short latency stretch reflexes to regulate greater levels of leg stiffness and RSI when hopping., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1) (27.2 kJ mol(-1)) for the carbinolamine formation reaction and 44.5 kcal mol(-1) (186 kJ mol(-1)) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1) (62.8 kJ mol(-1)) in the forward (formation) reaction and 19 kcal mol(-1) (79.5 kJ mol(-1)) for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N-terminal amine.

Human DNA polymerase kappa (pol κ) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N(2)-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N(2)-dG DNA-DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen ∼16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.

Oxidative damage to DNA is mainly repaired via base excision repair, a pathway that is catalyzed by DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1). While OGG1 has been implicated in maintaining genomic integrity and preventing tumorigenesis, we report a novel role for OGG1 in altering cellular and whole body energy homeostasis. OGG1-deficient (Ogg1(-/-)) mice have increased adiposity and hepatic steatosis following exposure to a high-fat diet (HFD), compared to wild-type (WT) animals. Ogg1(-/-) animals also have higher plasma insulin levels and impaired glucose tolerance upon HFD feeding, relative to WT counterparts. Analysis of energy expenditure revealed that HFD-fed Ogg1(-/-) mice have a higher resting VCO(2) and consequently, an increased respiratory quotient during the resting phase, indicating a preference for carbohydrate metabolism over fat oxidation in these mice. Additionally, microarray and quantitative PCR analyses revealed that key genes of fatty acid oxidation, including carnitine palmitoyl transferase-1, and the integral transcriptional co-activator Pgc-1α were significantly downregulated in Ogg1(-/-) livers. Multiple genes involved in TCA cycle metabolism were also significantly reduced in livers of Ogg1(-/-) mice. Furthermore, hepatic glycogen stores were diminished, and fasting plasma ketones were significantly reduced in Ogg1(-/-) mice. Collectively, these data indicate that OGG1 deficiency alters cellular substrate metabolism, favoring a fat sparing phenotype, that results in increased susceptibility to obesity and related pathologies in Ogg1(-/-) mice.

Aim: The purpose of this study was to determine from a range of vertical jump and rebound tests, which were the most suitable to measure different forms of stretch-shortening cycle function, and whether such tests could be used interchangeably., Methods: Two hundred and fifty male youths (age, 12.26 ± 2.94 years; body mass, 47.11 ± 16.91 kg; standing height, 152.98 ± 17.40 cm; and sitting height, 76.89 ± 9.32 cm) were tested for squat and countermovement jump height, reactive strength index (during a maximal hopping test), and leg stiffness (during a sub-maximal hopping test). Stepwise multiple regressions were used to examine the relationships between different measures of SSC function in youths., Results: Absolute leg stiffness was best predicted by body mass (r2=62%), however the explained variance was significantly reduced when normalized to leg length and body mass (r2=15.3%). Squat jump height best explained the total variance for reactive strength index (r2=53.9%), whilst countermovement and squat jump height were the best predictors of each other (r2=86%)., Conclusion: Results would suggest that the test protocols used in this study were representative of different forms of SSC performance. Coaches and athletes should take these findings into account when attempting to select the appropriate testing protocols to measure the correct SSC action.

The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Reaction of bifunctional electrophiles with DNA in the presence of peptides can result in DNA-peptide cross-links. In particular, the linkage can be formed in the major groove of DNA via the exocyclic amino group of adenine (N⁶-dA). We previously demonstrated that an A family human polymerase, Pol ν, can efficiently and accurately synthesize DNA past N⁶-dA-linked peptides. Based on these results, we hypothesized that another member of that family, Escherichia coli polymerase I (Pol I), may also be able to bypass these large major groove DNA lesions. To test this, oligodeoxynucleotides containing a site-specific N⁶-dA dodecylpeptide cross-link were created and utilized for in vitro DNA replication assays using E. coli DNA polymerases. The results showed that Pol I and Pol II could efficiently and accurately bypass this adduct, while Pol III replicase, Pol IV, and Pol V were strongly inhibited. In addition, cellular studies were conducted using E. coli strains that were either wild type or deficient in all three DNA damage-inducible polymerases, i.e., Pol II, Pol IV, and Pol V. When single-stranded DNA vectors containing a site-specific N⁶-dA dodecylpeptide cross-link were replicated in these strains, the efficiencies of replication were comparable, and in both strains, intracellular bypass of the lesion occurred in an error-free manner. Collectively, these findings demonstrate that despite its constrained active site, Pol I can catalyze DNA synthesis past N⁶-dA-linked peptide cross-links and is likely to play an essential role in cellular bypass of large major groove DNA lesions.

The γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine adduct (γ-OH-PdG) was introduced into 5'-d(GCTAGCXAGTCC)-3'·5'-d(GGACTCGCTAGC)-3' (X = γ-OH-PdG). In the presence of excess peptide KWKK, (13)C isotope-edited NMR revealed the formation of two spectroscopically distinct DNA-KWKK conjugates. These involved the reaction of the KWKK N-terminal amino group with the N(2)-dG propylaldehyde tautomer of the γ-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited (15)N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH(3), suggesting that they existed, in part, as imine (Schiff base) linkages. However, (13)C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNA-KWKK conjugates were predicted from potential energy minimization of model structures derived from the refined structure of the fully reduced cross-link [ Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. ( 2010 ) Biochemistry , 49 , 6155 -6164 ]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the S-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNA-KWKK conjugates thermally stabilized duplex DNA. However, the DNA-KWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 5'-CpX-3' sequence, the DNA-KWKK conjugates slowly converted to interstrand N(2)-dG:N(2)-dG DNA cross-links and ring-opened γ-OH-PdG derivatives over a period of weeks., (© 2011 American Chemical Society)

Although it is suggested that periods of naturally occurring accelerated adaptation may exist for various physical parameters, it would appear that no such evidence exists for stretch-shortening cycle (SSC) development. Two hundred and fifty male youths aged 7-17 years were tested for squat (SJ) and countermovement jump (CMJ) height, reactive strength index (RSI), and leg stiffness, with analyses of variance used to establish any significant between-group differences. Additionally, to ascertain the existence of periods of accelerated adaptation, inferences were made about the magnitudes of change between consecutive chronological age groups in relation to the smallest worthwhile change. The largest mean differences (±90% confidence limits) occurred between age groups 10 and 11 (G10-G11) for squat jump (SJ) height (21.61 ± 12.08-31.94%), CMJ height (20.80 ± 11.1-44.1%), and RSI (26.51 ± 11.07-44.10%); and between G12 and G13 for SJ (15.31 ± 7.47-23.73%) and CMJ (16.09 ± 7.50-25.38%) height. Negative mean differences occurred between G11 and G12 for SJ height (-1.32 ± -9.30 to 7.37%), CMJ jump height (-7.68 ± -15.15 to 0.45%) and RSI (-11.48 ± -22.21 to 0.74%); and between G10 and G11 for leg stiffness (-8.87 ± -18.85 to 2.34%). It would appear almost certain that windows of accelerated adaptation may exist for SJ and CMJ height and RSI in male youths; however, leg stiffness results would suggest that fast-SSC function may follow a different developmental trend.

An siRNA screen has identified several proteins throughout the base excision repair (BER) pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins.

UV light causes DNA damage in skin cells, leading to more than one million cases of non-melanoma skin cancer diagnosed annually in the United States. Although human cells possess a mechanism (nucleotide excision repair) to repair UV-induced DNA damage, mutagenesis still occurs when DNA is replicated before repair of these photoproducts. Although human cells have all the enzymes necessary to complete an alternate repair pathway, base excision repair (BER), they lack a DNA glycosylase that can initiate BER of dipyrimidine photoproducts. Certain prokaryotes and viruses produce pyrimidine dimer-specific DNA glycosylases (pdgs) that initiate BER of cyclobutane pyrimidine dimers (CPDs), the predominant UV-induced lesions. Such a pdg was identified in the Chlorella virus PBCV-1 and termed Cv-pdg. The Cv-pdg protein was engineered to contain a nuclear localization sequence (NLS) and a membrane permeabilization peptide (transcriptional transactivator, TAT). Here, we demonstrate that the Cv-pdg-NLS-TAT protein was delivered to repair-proficient keratinocytes and fibroblasts, and to a human skin model, where it rapidly initiated removal of CPDs. These data suggest a potential strategy for prevention of human skin cancer.

DNA-protein conjugates are potentially repaired via proteolytic digestion to DNA-peptide conjugates. The latter have been modeled with the amino-terminal lysine of the peptide KWKK conjugated via a trimethylene linkage to the N(2)-dG amine positioned in 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCGCTAGC)-3' (X = N(2)-dG-trimethylene link-KWKK). This linkage is a surrogate for the reversible linkage formed by the gamma-OH-1,N(2)-propanodeoxyguanosine (gamma-OH-PdG) adduct. This conjugated KWKK stabilizes the DNA. Amino acids K(26), W(27), K(28), and K(29) are in the minor groove. The W(27) indolyl group does not intercalate into the DNA. The G(7) N(2) amine and the K(26) N-terminal amine nitrogens are in the trans configuration with respect to the C(alpha) or C(gamma) of the trimethylene tether, respectively. The structure of this DNA-KWKK conjugate is discussed in the context of its biological processing.

UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links.

Trans-4-Hydroxynonenal (HNE) is a peroxidation product of omega-6 polyunsaturated fatty acids. Michael addition of HNE to deoxyguanosine yields four diastereomeric 1,N(2)-dG adducts. The adduct of (6S,8R,11S) stereochemistry forms interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence. It has been compared with the (6R,8S,11R) adduct, incorporated into 5'-d(GCTAGCXAGTCC)-3' . 5'-d(GGACTCGCTAGC)-3', containing the 5'-CpG-3' sequence (X = HNE-dG). Both adducts rearrange in DNA to N(2)-dG aldehydes. These aldehydes exist in equilibrium with diastereomeric cyclic hemiacetals, in which the latter predominate at equilibrium. These cyclic hemiacetals mask the aldehydes, explaining why DNA cross-linking is slow compared to related 1,N(2)-dG adducts formed by acrolein and crotonaldehyde. Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals are located within the minor groove. However, the (6S,8R,11S) cyclic hemiacetal orients in the 5'-direction, while the (6R,8S,11R) cyclic hemiacetal orients in the 3'-direction. The conformations of the diastereomeric N(2)-dG aldehydes, which are the reactive species involved in DNA cross-link formation, have been calculated using molecular mechanics methods. The (6S,8R,11S) aldehyde orients in the 5'-direction, while the (6R,8S,11R) aldehyde orients in the 3'-direction. This suggests a kinetic basis to explain, in part, why the (6S,8R,11S) HNE adduct forms interchain cross-links in the 5'-CpG-3' sequence, whereas (6R,8S,11R) HNE adduct does not. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease., (Environ. Mol. Mutagen., 2010. (c) 2010 Wiley-Liss, Inc.)

DNA polymerase nu (POLN or pol nu) is a newly discovered A family polymerase that generates a high error rate when incorporating nucleotides opposite dG; its translesion DNA synthesis (TLS) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions. In the current investigation, we describe a novel TLS substrate specificity of pol nu, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol nu catalyzed efficient and high fidelity TLS past peptides linked to N(6)-dA via a reduced Schiff base linkage with a gamma-hydroxypropano-dA. Additionally, pol nu could bypass DNA interstrand cross-links with linkage between N(6)-dAs in complementary DNA strands. However, the chemically identical DNA--peptide and DNA interstrand cross-links completely blocked pol nu when they were located in the minor groove via a N(2)-dG linkage. Furthermore, we showed that pol nu incorporated a nucleotide opposite the 1,N(6)-etheno-dA (epsilondA) in an error-free manner and (+)-trans-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-dA [(+)-BPDE-dA] in an error-prone manner, albeit with a greatly reduced capability. Collectively, these data suggest that although pol nu bypass capacity cannot be generalized to all major groove DNA adducts, this polymerase could be involved in TLS when genomic replication is blocked by extremely large major groove DNA lesions. In view of the recent observation that pol nu may have a role in cellular tolerance to DNA cross-linking agents, our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some DNA-protein and DNA-DNA cross-links.