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Several decades of research have identified Mcm10 hanging around the replisome making several critical contacts with a number of proteins but with no real disclosed function. Recently, the O'Donnell laboratory has been better able to map the interactions of Mcm10 with a larger Cdc45/GINS/MCM (CMG) unwinding complex placing it at the front of the replication fork. They have shown biochemically that Mcm10 has the impressive ability to strip off single-strand binding protein (RPA) and reanneal complementary DNA strands. This has major implications in controlling DNA unwinding speed as well as responding to various situations where fork reversal is needed. This work opens up a number of additional facets discussed here revolving around accessing the DNA junction for different molecular purposes within a crowded replisome. Abbreviations: alt-NHEJ: Alternative Nonhomologous End-Joining; CC: Coli-Coil motif; CMG: Cdc45/GINS/MCM2-7; CMGM: Cdc45/GINS/Mcm2-7/Mcm10; CPT: Camptothecin; CSB: Cockayne Syndrome Group B protein; CTD: C-Terminal Domain; DSB: Double-Strand Break; DSBR: Double-Strand Break Repair; dsDNA: Double-Stranded DNA; GINS: go-ichi-ni-san, Sld5-Psf1-Psf2-Psf3; HJ Dis: Holliday Junction dissolution; HJ Res: Holliday Junction resolution; HR: Homologous Recombination; ICL: Interstrand Cross-Link; ID: Internal Domain; MCM: Minichromosomal Maintenance; ND: Not Determined; NTD: N-Terminal Domain; PCNA: Proliferating Cell Nuclear Antigen; RPA: Replication Protein A; SA: Strand Annealing; SE: Strand Exchange; SEW: Steric Exclusion and Wrapping; ssDNA: Single-Stranded DNA; TCR: Transcription-Coupled Repair; TOP1: Topoisomerase.

Timely recruitment of DNA damage response proteins to sites of genomic structural lesions is very important for signaling mechanisms to activate appropriate cell cycle checkpoints but also repair the altered DNA sequence to suppress mutagenesis. The eukaryotic cell is characterized by a complex cadre of players and pathways to ensure genomic stability in the face of replication stress or outright genomic insult by endogenous metabolites or environmental agents. Among the key performers are molecular motor DNA unwinding enzymes known as helicases that sense genomic perturbations and separate structured DNA strands so that replacement of a damaged base or sugar-phosphate backbone lesion can occur efficiently. Mutations in the BLM gene encoding the DNA helicase BLM leads to a rare chromosomal instability disorder known as Bloom's syndrome. In a recent paper by the Sengupta lab, BLM's role in the correction of double-strand breaks (DSB), a particularly dangerous form of DNA damage, was investigated. Adding to the complexity, BLM appears to be a key ringmaster of DSB repair as it acts both positively and negatively to regulate correction pathways of high or low fidelity. The FANCJ DNA helicase, mutated in another chromosomal instability disorder known as Fanconi Anemia, is an important player that likely coordinates with BLM in the balancing act. Further studies to dissect the roles of DNA helicases like FANCJ and BLM in DSB repair are warranted.

Figuring out what is wrong in Fanconi anemia (FA) patient cells is critical to understanding the contributions of the FA pathway to DNA repair and tumor suppression. Although FA patients exhibit a wide range of disease manifestation as well as severity (asymptomatic to congenital abnormalities, bone marrow failure, and cancer), cells from FA patients share underlying defects in their ability to process DNA lesions that interfere with DNA replication. In particular, FA cells are very sensitive to agents that induce DNA interstrand crosslinks (ICLs). The cause of this pronounced ICL sensitivity is not fully understood, but has been linked to the aberrant activation of DNA damage repair proteins, checkpoints and pathways. Thus, regulation of these responses through coordination of repair processing at stalled replication forks is an essential function of the FA pathway. Here, we briefly summarize some of the aberrant DNA damage responses contributing to defects in FA cells, and detail the newly-identified relationship between FA and the mismatch repair protein, MSH2. Understanding the contribution of MSH2 and/or other proteins to the replication problem in FA cells will be key to assessing therapeutic options to improve the health of FA patients. Moreover, loss of these factors, if linked to improved replication, could be a key event in the progression of FA cells to cancer cells. Likewise, loss of these factors could synergize to enhance tumorigenesis or confer chemoresistance in tumors defective in FA-BRCA pathway proteins and provide a basis for biomarkers for disease progression and response.

Five members of the RecQ subfamily of DEx-H-containing DNA helicases have been identified in both human and mouse, and mutations in BLM, WRN, and RECQ4 are associated with human diseases of premature aging, cancer, and chromosomal instability. Although a genetic disease has not been linked to RECQ1 mutations, RECQ1 helicase is the most highly expressed of the human RecQ helicases, suggesting an important role in cellular DNA metabolism. Recent advances have elucidated a unique role of RECQ1 to suppress genomic instability. Embryonic fibroblasts from RECQ1-deficient mice displayed aneuploidy, chromosomal instability, and increased load of DNA damage.(1) Acute depletion of human RECQ1 renders cells sensitive to DNA damage and results in spontaneous gamma-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks.(2) Consistent with a role in DNA repair, RECQ1 relocalizes to irradiation-induced nuclear foci and associates with chromatin.(2) RECQ1 catalytic activities(3) and interactions with DNA repair proteins(2,4,5) are likely to be important for its molecular functions in genome homeostasis. Collectively, these studies provide the first evidence for an important role of RECQ1 to confer chromosomal stability that is unique from that of other RecQ helicases and suggest its potential involvement in tumorigenesis.

Post-translational modifications of target DNA repair proteins may help to regulate the mechanism of DNA damage correction. A good analogy is an automobile tune-up necessary to make a car operate a...

Bloom’s syndrome (BS) is a rare autosomal recessive genetic disorder characterized by predisposition to a wide variety of cancers observed in the normal population.1 The BLM gene defective in BS encodes a RecQ DNA helicase (BLM) that is important for genomic stability by suppressing sister chromatid exchanges (SCE) that arise during homologous recombination (HR).2 In fact, SCE frequency of patient cells is used for clinical diagnosis of BS. BLM helicase is believed to suppress SCEs by channeling DNA molecules away from pathways leading to crossover products through its DNA unwinding function and interaction with protein partners (e.g., human Topoisomerase IIIα).3 Targeting DNA helicases for therapeutic purposes has attracted interest with the discovery of other DNA repair inhibitors, highlighted by poly(ADP)ribosylase (PARP) inhibitors used in synthetic lethal approaches to attenuate carcinogenesis in HR-defective BRCA1/2-deficient tumors.4 Small molecules (< 800 Daltons) can penetrate cell membranes and represent a potentially suitable class of compounds for therapeutic use, such as anti-cancer drugs. In the January 24, 2013 issue of Chemistry and Biology, Nguyen et al. reported their discovery of a small molecule inhibitor of BLM helicase.5 From a high throughput screen of a chemical compound library and medicinal chemistry optimization, a small molecule (ML216) was identified that inhibited BLM helicase activity on a forked duplex DNA substrate in vitro (IC50 ~3 μM) by preventing BLM binding to DNA.5 Cultured human fibroblasts exposed to ML216 (50 μM) displayed reduced proliferation, a statistically significant increase in SCE frequency, and elevated sensitivity to aphidicolin, an inhibitor of replicative DNA polymerases. The specificity for ML216 targeting BLM in cell-based experiments was suggested because BLM-deficient cells were resistant to the phenotypic effects of ML216. The BLM helicase inhibitor discovery may provide a new strategy for understanding molecular functions of BLM required for its role in chromosomal stability, and also potential development of a new class of chemotherapy drugs to treat tumors which rely heavily on BLM for proliferation. From a biochemist’s perspective, it is intriguing that ML216 potently inhibited BLM unwinding of a forked DNA duplex substrate, but only modestly affected unwinding of other DNA substrates (G-quadruplex, Holliday Junction, or plasmid-based D-loop) at much higher concentrations of drug.5 The specificity of ML216 (and conceivably other helicase inhibitors) may allow an experimental approach to dissect molecular requirements of the helicase for its role(s) in genome stability. Although ML216 inhibited unwinding by the sequence-related BLM and WRN helicases similarly in vitro, the apparent dependence on BLM for ML216 to exert its biological effects in human cells suggests BLM specificity for the drug’s mechanism of action in vivo. A co-crystal structure of BLM in complex with inhibitor would be informative. Cellular cues in vivo may induce a specific conformation of WRN that makes it resistant to ML216. Direct or water-mediated contacts of the small molecule with poorly conserved amino acid residues of BLM that are distal in the primary structure but proximal in the tertiary structure may be critical for drug action. Other studies reporting pharmacological inhibition of DNA repair protein function have also shown a dependence on target protein for the small molecule’s cellular effect. An inhibitor of WRN helicase (NSC 19630) was discovered that inhibited proliferation and induced DNA damage and apoptosis in human cancer cells in a WRN-dependent manner.6 Although the mechanism of action whereby NSC 19630 interferes with critical function(s) of WRN at the cellular level is unknown, there are several avenues to investigate. The WRN-inhibitor drug complex may prevent WRN from interacting favorably with its protein partners or cause formation of a static protein-DNA complex that is deleterious to normal biological DNA transactions. Since NSC 19630 exerted only a marginal effect on DNA-dependent WRN ATPase or exonuclease activity in vitro at very high drug concentrations,6 WRN inhibitor is likely to operate by a mechanism distinct from that of the BLM inhibitor which adversely affected BLM DNA binding and DNA-dependent ATPase activity at relatively low drug concentrations.5 Our current hypothesis is that the biological effects of NSC 19630 may at least partly reflect an inactive WRN helicase-drug complex trapped on DNA repair or replication intermediates. Further studies will be necessary to determine if this is the case. However, a recent study of clinical PARP inhibitors that operate in a PARP-dependent manner hinted at a provocative scenario. Small molecule inhibition of PARP1 or PARP2 became more cytotoxic than genetic depletion of PARP by causing PARP to become trapped on DNA at damaged sites.7 This finding suggests a reasonable mechanism for a class of DNA helicase inhibitors (like NSC 19630), but more research is necessary. Understanding the mechanisms of DNA repair inhibitors has potential clinical significance. Chemo- and radio-therapy approaches to combat cancer are largely based on introducing DNA damage leading to double strand breaks (DSB). Recently, a small molecule inhibitor (SCR7) of DNA Ligase IV responsible for nonhomologous end-joining (NHEJ) was discovered and found to inhibit NHEJ in a Ligase IV-dependent manner,8 reminiscent of the helicase and PARP inhibitors discussed above. Importantly, SCR7 impeded tumor progression in mouse models.8 Hopefully, further research and clinical applications for helicase inhibitors will prove to be promising.

Comment on: WRN helicase defective in the premature aging disorder Werner Syndrome genetically interacts with topoisomerase 3 and restores the top3 slowgrowth phenotype of sgs1 top3. Monika Aggarwal, Robert M. Brosh, Jr. Aging 2009; 1:219-233

The complex process of semi-conservative DNA replication involves a mechanism whereby the leading and lagging strands with opposite polarity serve as templates for concerted synthesis of complementary base pairs.1 Lagging-strand synthesis creates discontinuous Okazaki fragments that require timely processing of the 5′ flaps, so that adjacent nascent DNA strands are ligated together to insure genomic stability. While the genetic and molecular requirements of Okazaki fragment maturation have been studied in much detail, the precise temporal and spatial relationship of lagging-strand processing to sister chromatid cohesion remains unclear.2 The newly replicated daughter duplex DNA molecules (i.e., the sister chromatids) become tethered during DNA replication and remain paired in order to permit proper segregation of the chromosomes to respective poles during mitosis and nuclear division. Elegant genetic studies in yeast have implicated posttranslational modification of cohesins (specialized protein complexes responsible for tethering sister pairs) by Ctf7/Eco1 acetylase as a key regulatory step in the process, enabling cohesins to perform their function in capturing the newly synthesized sister chromatids. Previous work suggested that genetic and physical interactions among the yeast acetyltransferase Ctf7/Eco1, helicase Chl1, Flap Endonuclease (Fen1) and accessory replication factors [e.g., RFC (clamp loader) and PCNA (clamp)] play an integral role in cohesion establishment. Based on these pieces of evidence, several models to explain the relationship between replication fork dynamics and sister chromatid cohesion have been proposed; however, our understanding of the precise timing of cohesin acetylation and the passage of the replication fork machinery has remained murky at best. Given the importance of proper chromosome segregation for chromosomal stability and the suppression of developmental disorders and tumorigenesis, a comprehensive understanding of the molecular acrobatics involved in sister chromatid cohesion is highly important. In a recent study, the temporal relationship between sister chromatid establishment and lagging-strand synthesis was illuminated.3 The authors have elucidated the link between the catalytic functions of DNA unwinding, flap processing and acetylation, which supports a model of cohesion deposition and establishment that occurs after the passage of the replication fork, similar to how genomic DNA becomes chromatinized. This is a significant advance from an earlier and very popular model of sister chromatid cohesion predicted that Ctf7/Eco1 acetylated cohesin proteins before the encounter by the DNA replication fork, which was thought to permit fork progression and the proper cohesion state for sister chromatid tethering (for review, see ref. 2). Instead, the genetic evidence presented by the Skibbens lab supports a model whereby cohesion establishment is temporally coupled to lagging-strand processing.3 In support of the genetic proof, Rudra and Skibbens went on to show that both Ctf7/Eco1 and Chl1 are associated with the lagging-strand processing nuclease Fen1. Altogether, the experimental results implicate a post-fork establishment model that is analogous to how histone protein complexes are deposited onto newly synthesized sister chromatids and become posttranslationally modified to confer epigenetic status. The discovery from the Skibbens lab that cohesion establishment is closely orchestrated with Okazaki fragment processing prompts a new line of inquiry about the control of flap processing by acetylation and its dual purpose for proper sister chromatid cohesion and replication fidelity in eukaryotes (Fig. 1). The catalytic activity of human FEN-14,5 and a functionally related endonuclease known as Dna24 have been shown to be modulated by p300 acetylation, which suggested a model for creating long flap intermediates to promote genomic stability and suppress mutagenesis. Given evidence that ChlR1 is implicated in the genetic disorder Warsaw Breakage syndrome and that the human homolog of yeast Chl16 interacts with the RFC complex and Fen1,7 it will be informative to determine if acetyltransferases such as the human orthologs Esco1 and Esco2, the latter mutated in the cohesinopathy Roberts syndrome,8 and perhaps other acetyltransferases (e.g., p300) are master regulators of lagging-strand synthesis that not only affect replication fidelity and genomic stability, but also sister chromatid cohesion. Coordination of sister chromatid cohesion establishment with lagging strand synthesis may also involve replication fork stabilization by the Timeless-Tipin protein complex implicated in replication checkpoint.9 Defects in the efficient coupling of lagging-strand synthesis to sister chromatid cohesion may contribute to the chromosomal instability characteristic of age-related diseases and cancer. Figure 1. Interplay between acetylation, replication fork dynamics and cohesion establishment important for chromosomal integrity.