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Intrinsically disordered proteins (IDPs) lack unique 3D structures under native conditions and yet retain critical functions. Recycling of RNA Polymerase II after transcription is promoted by an interaction between the winged helix domain of RAP74, a component of the general transcription factor IIF (TFIIF), and the C-terminus of the TFIIF-associating CTD phosphatase (FCP1). Sixteen residues from the C-terminus of FCP1 form an α-helix in the complex, but the protein is otherwise agreed in the literature to be intrinsically disordered. Here we show through CD and recently developed carbon-detected NMR that, although FCP1 is intrinsically disordered, the above 16 residues composing the RAP74 binding surface form nascent α-helical structure in the unbound state. We further show retention of general FCP1 disorder and the nascent helical content in HeLa extract, establishing cellular relevance. The conformational bias observed leads to a mechanistic proposal for FCP1's transition from a disordered ensemble to an ordered conformation upon binding., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Errol Friedberg suggested that I write a biographical account of the work carried out in my lab for the Historical Reflections section of the DNA Repair. Although I started out studying meiotic recombination, I have spent much of the last four and a half decades focused on trying to understand the mechanism underlying induced mutagenesis, which led me into what was eventually called DNA damage tolerance, the process that facilitates the resumption of replication when replicases are stalled at sites of DNA template damage. The following account highlights some of our work that contributed to an understanding of the mechanisms underlying these activities, carried out by the RAD6 pathway, my main preoccupation over this period.

Mismatch-repair (MMR) systems suppress mutation via correction of DNA replication errors (base-mispairs) and responses to mutagenic DNA lesions. Selective binding of mismatched or damaged DNA by MutS-homolog proteins-bacterial MutS, eukaryotic MSH2.MSH6 (MutSalpha) and MSH2.MSH3-initiates mismatch-correction pathways and responses to lesions, and may cumulatively increase discrimination at downstream steps. MutS-homolog binding selectivity and the well-known but poorly understood effects of DNA-sequence contexts on recognition may thus be primary determinants of MMR specificity and efficiency. MMR processes that modulate UV mutagenesis might begin with selective binding by MutS homologs of "mismatched" T[CPD]T/AG and T[6--4]T/AG photoproducts, reported previously for hMutSalpha and described here for E. coli MutS protein. If MMR suppresses UV mutagenesis by acting directly on pre-mutagenic products of replicative bypass, mismatched photoproducts should be recognized in most DNA-sequence contexts. In three of four contexts tested here (three substantially different), T[CPD]T/AG was bound only slightly better by MutS than was T[CPD]T/AA or homoduplex DNA; only one of two contexts tested promoted selective binding of T[6--4]T/AG. Although the T:G pairs in T[CPD]T/AG and T/G both adopt wobble conformations, MutS bound T/G well in all contexts (K(1/2) 2.1--2.9 nM). Thus, MutS appears to select the two mismatches by different mechanisms. NMR analyses elsewhere suggest that in the (highly distorted) T[6--4]T/AG a forked H-bond between O2 of the 3' thymine and the ring 1-imino and exocyclic 2-amino guanine protons stabilizes a novel planar structure not possible in T[6--4]T/AA. Replacement of G by purines lacking one (inosine, 2-aminopurine) or both (nebularine) protons markedly reduced or eliminated selective MutS binding, as predicted. Previous studies and the work here, taken together, suggest that in only about half of DNA sequence contexts could MutS (and presumably MutSalpha) selectively bind mismatched UV photoproducts and directly suppress UV mutagenesis.

In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not possible to demonstrate that the decrease in mutagenesis reflected decreased hRev3 protein. However, the conclusion is supported by the fact that in a similar study with a strain expressing a high level of antisense hREV1, a very similar result was obtained, i.e. UV or BPDE mutagenesis was virtually eliminated.

The majority of both spontaneous and DNA damage-induced mutations in eukaryotes result from replication processes in which DNA polymerase zeta (Polzeta) and Rev1 protein (Rev1p) play major roles. Understanding these roles is likely to provide information relevant to the origin of genetic diseases, such as cancer, and may provide new insights for their prevention. DNA Polzeta also appears to be involved in the somatic hypermutability that occurs during development of the immune response. The results from a variety of genetic and enzymological investigations have started to delineate the cellular roles of these enzymes, but a number of important issues have not yet been resolved and much remains to be learned. Questions concerning the possible existence of other subunits to these enzymes, of their possible association with one another or with other proteins, of the nature of their enzymatic activities and of the relative roles played by these and other DNA polymerases in the bypass of different kinds of DNA damage, require further investigation. Finally, very little is known about the way these enzymes are regulated and brought into play when needed.

DNA replication in eucaryotic cells involves a variety of proteins which synthesize the leading and lagging strands in an asymmetric coordinated manner. To analyse the effect of this asymmetry on the translesion synthesis of UV-induced lesions, we have incubated SV40 origin-containing plasmids with a unique site-specific cis, syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct on either the leading or lagging strand template with DNA replication-competent extracts made from human HeLa cells. Two dimensional agarose gel electrophoresis analyses revealed a strong blockage of fork progression only when the UV lesion is located on the leading strand template. Because DNA helicases are responsible for unwinding duplex DNA ahead of the fork and are then the first component to encounter any potential lesion, we tested the effect of these single photoproducts on the unwinding activity of the SV40 T antigen, the major helicase in our in vitro replication assay. We showed that the activity of the SV40 T-antigen helicase is not inhibited by UV-induced DNA lesions in double-stranded DNA substrate.

Abasic sites are particularly important in mutation research because they are frequently the ultimate lesion in chemical mutagenesis, and because they are believed to be a paradigm for non-pairing lesions. Although preferential insertion of dAMP ("A-rule") opposite the lesion has been observed in almost all previous studies with other organisms, we find that in budding yeast, Saccharomyces cerevisiae, the preferred nucleotide is dCMP, suggesting that yeast has a "C-rule", at least with respect to the vector constructs used. These constructs contained a single abasic site specifically located within a 28 nucleotide single-stranded region in an otherwise duplex vector. Nucleotide insertions were determined by sequence analysis of replicated vectors taken from a random set of yeast transformants. In three different sequence contexts, the frequencies of dCMP and dAMP insertion were 83% and 13%, 62% and 31%, and 85% and 8%, respectively. A similar bias in favor of cytosine insertion was found using vectors that were entirely single-stranded. However, a preference for dAMP insertion was found when Escherichia coli, rather than yeast, was transfected with samples of the same gapped duplex vector DNA. Preferential insertion of dCMP is not likely to have arisen by previously proposed mechanisms, but is also unlikely to have occurred by a primer/template misalignment mechanism, in which a nearby template guanine directs the insertion of cytosine. Predominant dCMP insertion was observed even when template guanine bases were excluded from a region extending 19 nucleotides 5', and 13 nucleotides 3', to the abasic site.

The thymine-cytosine pyrimidine-pyrimidone (6-4) adduct has variously been predicted to be among the most and among the least mutagenic of the ultraviolet light photoproducts. We have therefore investigated the frequency and accuracy of DNA replication past this lesion, using a single-stranded M13mp7-based vector with a uniquely located example of this lesion transfected into SOS-induced and uninduced cells of a uvr A6 strain of Escherichia coli. Both the UVC T-C (6-4) adduct and its Dewar valence (UVB) photoisomer were studied. Random samples from non-selective collections of progeny phage were sequenced to determine the nature of the replication events that occurred at or near the site of template damage under SOS conditions. The UVC (6-4) adduct was found to be much less mutagenic than its T-T counterpart, but still much more mutagenic than a cyclobutane dimer; 34% (71 out of 206) of all bypass events yielded mutations, of which all were targeted and 80% (57 out of 71) were 3' C-->T transitions. The Dewar valence photoisomer exhibited reduced specificity and enhanced mutagenicity; 79% (183 out of 233) of the phage progeny were mutants, of which all but one were targeted and 45% (83 out of 183) were 3' C-->T transitions. For the most part, these results are consistent with a model postulating base-pairing between the pyrimidinone (of either the C or T variety) and guanine, via hydrogen bonds at N-3 and O-2 in the UVC, but not the Dewar, isomer. The occurrence of the 3' C-->T transitions, not predicted by this model, shows however that the absence of a methyl group at C-5 also has a significant influence on mutation induction. Both isomers were efficient blocks to replication; less than 1% of these vectors could be replicated in uninduced cells. Following SOS induction the frequency of bypass increased to 24.5% and 12.5% for the UVC and the Dewar isomers, respectively.

Data from experiments with single-stranded vectors that carry a site-specific cyclobutane dimer, pyrimidine (6-4) pyrimidone adduct, or abasic lesion, replicated in either E. coli or, in some cases, bakers' yeast, Saccharomyces cerevisiae, are used to examine two questions: (i) what factors are responsible for the lesion's mutagenicity? and (ii) what are the relative contributions of different photoproducts to the spectrum of UV-induced mutations? With respect to the first question, we suggest that the structure of the mutagen-modified template itself largely determines the kinds of mutations induced, but the relative frequencies of these mutations, the error frequency, and the bypass frequency are strongly dependent on the particular organism studied. With respect to the second question, we suggest that cyclobutane dimers may be responsible for most of the mutations in slowly replicating genomes because of the deamination of cytosine, and that the T-T, and to a lesser extent the T-C, (6-4) adducts play a greater role in the UV mutagenesis of quickly replicating viruses, such as M13 and lambda phage.

Comparisons between the 60Co gamma-ray survival curves of diploid strains of the yeast Saccharomyces cerevisiae that are homozygous for two non-allelic radiation-sensitive mutations and the corresponding single-mutant diploids suggest that there are two main types of repair of ionizing radiation damage in this organism. The first, which is defined by the rad52 epistasis group, depends on the activities of the RAD50 through RAD57 genes and is responsible for repairing the larger amount of lethal damage. Previous work [22] shows that this type of repair is essentially error-free. The second, defined by the rad6 epistasis group, depends on the activities of the RAD6, RAD9, RAD18, REV1 and REV3 genes and repairs a smaller, though still substantial, amount of lethal damage. It is also responsible for induced mutagenesis [22, 23]. Data for survival and mutation induction after irradiation in air and partial anoxia show that oxygen-dependent damage can be repaired by either of these two pathways. They also show similar oxygen-enhancement ratios for survival and mutagenesis.

We have isolated and characterized a mutant of baker's yeast, Saccharomyces cerevisiae, carrying the new mutation, ngs1, which is sensitive to the toxic effects of monofunctional alkylating agents, but normal with respect to 254-nm ultraviolet light sensitivity. ngs1 mutants exhibited more or less normal reversion frequencies for his1-7 and ilv1-92 induced by each of these mutagens. The various sensitivities associated with ngs1 cosegregated and have been shown to be the result of a lesion in a single nuclear gene. Extracts of ngs1 and NGS1+ strains contained approximately equal levels of an activity that removes 3-methyladenine (3MA) and 7-methylguanine (7MG) from DNA in vitro. The mutation also depressed sporulation.

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

We have analyzed the nucleotide sequence changes responsible for mutations from lacIs to lacI- induced in ultraviolet light-irradiated, excision-deficient cells. Irradiated cells were either used as donors in the conjugational transfer of an F' lacIs plasmid to SOS-induced, excision-deficient recipients or allowed to continue vegetative growth. Although the types and proportions of premutagenic lesions are likely to have been very similar in these two circumstances, analysis of the sequence data shows that different spectra of mutations were induced. In vegetative cells there were about equal numbers of transitions and transversions, but transitions outnumbered transversions by about three to one in exconjugants. About 90% of the single nucleotide substitutions could be assigned to a bipyrimidine target sequence in both sets of data, but they differed with respect to the location of the substitution: more or less equal numbers were found at the 3' and 5' sites of the probable bipyrimidine target in vegetative cells, but in exconjugants over 80% were at the 3' site. It is also possible that mutations were targeted more commonly at T-C sequences in exconjugants than in vegetative cells, but the evidence for this is less secure. We conclude that these results reflect some dissimilarity between vegetative cells and exconjugants in the way damaged DNA is replicated or lesions tolerated, but the particular features of these processes responsible for the different mutational spectra have not yet been identified.

Up to a quarter or more of the normal yield of lacI- mutations could be induced by ultraviolet light in a uvrA6 umuC122:: Tn5 strain if they were detected by plating on 5-bromo-4-chloro-3-indolyl-beta-D-galactoside medium, where all surviving cells can form colonies. Using phenyl beta-D-galactoside selection, which curtails post-irradiation growth, only low yields of mutations were induced. Nucleotide sequence analysis of 134 spontaneous and 145 ultraviolet light-induced mutations shows that broadly similar kinds of mutations were induced in the umuC mutant and its uvrA6 umuC+ counterpart. In particular, these data offer no reason for believing that most of the mutations induced in the umuC mutant were other than normal, targeted events. We conclude that UmuC function, rather than being essential, facilitates recovery and specifically, following the model of Bridges & Woodgate, that it facilitates the prompt resumption of chain elongation.

Retinyl-15-(14)C palmitate-9,10-(3)H was fed to rats in order to study hydrolysis and reesterification of this ester during digestion, absorption, transportation, and storage. After administration there was a progressive increase in the (14)C/(3)H ratio of the retinyl esters as they moved from intestinal contents to intestinal mucosa, lymph, and liver, which indicates that repeated hydrolysis and reesterification occur during the digestion and assimilation of this ester.