Error-prone Postreplication Repair Research Articles

The Preference for Error-Free or Error-Prone Postreplication Repair in Saccharomyces cerevisiae Exposed to Low-Dose Methyl Methanesulfonate Is Cell Cycle Dependent

Cells employ error-free or error-prone postreplication repair (PRR) processes to tolerate DNA damage. Here, we present a genome-wide screen for sensitivity to 0.001% methyl methanesulfonate (MMS). This relatively low dose is of particular interest because wild-type cells exhibit no discernible phenotypes in response to treatment, yet PRR mutants are unique among repair mutants in their exquisite sensitivity to 0.001% MMS; thus, low-dose MMS treatment provides a distinctive opportunity to study postreplication repair processes. We show that upon exposure to low-dose MMS, a PRR-defective rad18Δ mutant stalls into a lengthy G2 arrest associated with the accumulation of single-stranded DNA (ssDNA) gaps. Consistent with previous results following UV-induced damage, reactivation of Rad18, even after prolonged G2 arrest, restores viability and genome integrity. We further show that PRR pathway preference in 0.001% MMS depends on timing and context; cells preferentially employ the error-free pathway in S phase and do not require MEC1-dependent checkpoint activation for survival. However, when PRR is restricted to the G2 phase, cells utilize REV3-dependent translesion synthesis, which requires a MEC1-dependent delay and results in significant hypermutability.

Structural Basis of Recruitment of DNA Polymerase ζ by Interaction between REV1 and REV7 Proteins

REV1, REV3, and REV7 are pivotal proteins in translesion DNA synthesis, which allows DNA synthesis even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases and function as inserter and extender polymerases in this process, respectively. REV7 interacts with both REV1 and REV3, acting as an adaptor that functionally links the two, although the structural basis of this collaboration remains unclear. Here, we show the crystal structure of the ternary complex, composed of the C-terminal domain of human REV1, REV7, and a REV3 fragment. The REV1 C-terminal domain adopts a four-helix bundle that interacts with REV7. A linker region between helices 2 and 3, which is conserved among mammals, interacts with the β-sheet of REV7. Remarkably, the REV7-binding interface is distinct from the binding site of DNA polymerase η or κ. Thus, the REV1 C-terminal domain might facilitate polymerase switching by providing a scaffold for both inserter and extender polymerases to bind. Our structure reveals the basis of DNA polymerase ζ (a complex of REV3 and REV7) recruitment to the stalled replication fork and provides insight into the mechanism of polymerase switching.

Multifaceted Recognition of Vertebrate Rev1 by Translesion Polymerases ζ and κ

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.

Identification of pathways controlling DNA damage induced mutation in Saccharomyces cerevisiae

Mutation in response to most types of DNA damage is thought to be mediated by the error-prone sub-branch of post-replication repair and the associated translesion synthesis polymerases. To further understand the mutagenic response to DNA damage, we screened a collection of 4848 haploid gene deletion strains of Saccharomyces cerevisiae for decreased damage-induced mutation of the CAN1 gene. Through extensive quantitative validation of the strains identified by the screen, we identified ten genes, which included error-prone post-replication repair genes known to be involved in induced mutation, as well as two additional genes, FYV6 and RNR4. We demonstrate that FYV6 and RNR4 are epistatic with respect to induced mutation, and that they function, at least partially, independently of post-replication repair. This pathway of induced mutation appears to be mediated by an increase in dNTP levels that facilitates lesion bypass by the replicative polymerase Polδ, and it is as important as error-prone post-replication repair in the case of UV- and MMS-induced mutation, but solely responsible for EMS-induced mutation. We show that Rnr4/Polδ-induced mutation is efficiently inhibited by hydroxyurea, a small molecule inhibitor of ribonucleotide reductase, suggesting that if similar pathways exist in human cells, intervention in some forms of mutation may be possible.

Studies on mutagen-sensitive strains of Drosophila melanogaster: IX. Modification of genetic damage induced by X-irradiation of spermatozoa in N 2, air or O 2 by 4 autosomal repair-deficient mutants

The influence of defects in DNA repair on the recovery of X-ray-induced genetic damage in spermatozoa of Drosophila melanogaster was studied. Basc males were irradiated in N 2, air or O 2 and mated to females of 4 repair-deficient mutant types: mus(2)201 D1 (excision repair deficient), mus(3)306 D1 (excision repair deficient), mus(3)302 D1 and mus(3)308 D2 (both excision repair and post-replication repair deficient). The frequencies of sex-lined recessive lethals and of autosomal translocations in the F 1 progeny were determined following standard genetic procedures. The responses in the different crosses with repair-deficient females were compared to those with repair-proficient mei + females (maternal effects). The main findings are the following: (1) with excision repair-deficient females the frequencies of spontaneous recessive lethals tend to be higher than with mei + females; (2) with excision repair-deficient females the frequencies of recessive lethals induced in N 2 and air and often in O 2 are higher than with mei + females; (3) with post-replication repair-deficient mutants a maternal effect is found for X-ray-induced translocations - both increases and decreases occur depending on the specific mutant type. The data are explained as follows: excision repair deficiencies cause the processing of primary lesions to be diverted from the error-free excision repair to the error-prone post-replication repair pathways. This results in enhanced mutational yields. After irradiation in O 2 secondary effects cause selective elimination of potential recessive lethals in those mutants that exhibit lowered fertility ( mei-9, mus-302). Therefore those mutants have no differential maternal effect on the recovery of recessive lethals after irradiation in O 2. The changes in translocation yield with post-replication repair deficiencies are thought to be the result of defects in the repair of DNA breaks. These defects might cause the post-replication repair deficiency too. The group of post-replication repair mutants is heterogenous. The mutants with low fertility seem to cause decreased translocation frequencies by selective elimination through dominant lethality. The other mutants increase the frequencies.

Enhancement of excision-repair efficiency by conditioned medium from density-inhibited cultures in V79 Chinese hamster cells: evidence for excision repair as an error-free repair process.

Conditioned medium from density-inhibited V79 Chinese hamster cell cultures, given as a post-treatment to UV-irradiated homologous cells, was demonstrated to reduce the lethal action of ultraviolet light by temporarily blocking DNA replication. Since the increased survival was not affected by various non-toxic concentrations of caffeine, such protective effect would be attributable to the prolonged intervention of excision repair before DNA replication during the post-treatment period. The influence of conditioned medium on the UV-induced mutation at the ouabain-resistance locus was also examined and a significant decrease in mutation frequency was noted. The observed reduction in killing and mutation as a result of post-incubation in conditioned medium, which delays DNA replication, would be interpreted as evidence that conditioned medium provides a longer period of time for an error-free excision-repair process, leaving lesion in DNA available for error-prone post-replication repair.

Effects of a short heat pulse on phenomena related to DNA repair in ultraviolet-irradiated Escherichia coli.

Mutational synergism between heat and UV light in Escherichia coli was examined for evidence supporting various possible synergistic mechanisms. UV and heat lethality were only additive or slightly synergistic after 30 sec at 50 degrees C. The order of treatment did not affect the mutational synergism. Synergism is not peculiar to induction of mutation from streptomycin sensitivity to resistance. Neither DNA breakage nor alteration of post-replication repair rates was seen after a 5 min 50 degrees C heat pulse. Synergism was observed with temperatures as low as 43 degrees C. A strain deficient in excision repair but not post-replication repair exhibited mutational synergism, suggesting that synergism may involve the differential heat alteration of the error-prone post-replication repair process.

Partial suppression of the LexA phenotype by mutations ( rnm) which restore ultraviolet resistance but not ultraviolet mulability to Escherichia coli B/r uvrA lexA

In Escherichia coli, lexA mutations eliminate expression of UV-inducible functions, causing pleiotropic effects which include sensitivity to ultraviolet (UV) light and loss of UV mutability. Selection for UV resistance, after 5-bromouracil (BU) treatment of E. coli B/r uvr A lexA-102, has yielded derivatives more resistant than lexA but still refractory to UV mutagenesis. The mutation responsible for the UV-resistant UV-nonmutable phenotype ( rnm) is cotransducible with malB to about the same extent as is lexA-102 and is tightly linked to lexA-102 in at least one strain. The rnm mutation may therefore be an intragenic partial suppressor of the LexA phenotype. In addition to increased UV resistance and lack of UV mutability, rnm strains show improved ability to perform postreplication repair and to control postirradiation DNA degration compared to the lexA parent. We ascribe the properties of rnm mutants to their having reacquired control of Exonuclease V activity without having reacquired UV-inducible error-prone postreplication repair. We relate our results to current interpretations of UV mutagenesis and to models of coordinate regulation of UV-inducible functions.

Caffeine inhibits cell transformation by 4-nitroquinoline-1-oxide.

SOMATIC mutation has been considered a likely initiating step in chemical carcinogenesis1–3, chiefly because of the close correlation between the mutagenic and carcinogenic activity of various chemicals4–6. Study of cells from patients with disease predisposing to a high incidence of cancer, such as xeroderma pigmentosum and Fanconi's anaemia, has suggested that defective DNA repair is correlated with this predisposition to malignant transformation. When cells are transformed by 3-methylcholanthrene7, 4-nitroquinoline-1-oxide (4NQO)8, or X irradiation9–11, division is necessary soon after treatment to fix the transformation: if treated cells are unable to grow the transformation is not fixed. These results could be explained if DNA damage induced by a carcinogen is converted into a stable and replicable structural change only by means of DNA replication before the intervention of error-free repair mechanisms. The model of mutagenesis12–14 based on evidence obtained with micro-organisms suggests that errors introduced by an error-prone postreplication repair mechanism are the principal source of mutation. Furthermore, caffeine inhibits “postreplication repair” (presumably an error-prone mechanism) but not excision repair (presumably error-free) in rodent cells15–19. I now report that caffeine reduces the transformation frequency of mouse cells treated with 4NQO. This supports the somatic mutation theory of cell transformation.

Indirect mutagenesis in phage lambda by ultraviolet preirradiation of host bacteria

Mutations from “wild-type” to “clear” can be indirectly induced in unirradiated phage λc+ by ultraviolet preirradiation of host bacteria alone. Indirect mutagenesis to λc was not detected previously because the induced mutations occur in only small fractions of the offspring. The fluctuation test showed that the sizes of indirectly induced mutant clones were distributed from size one up to about half of the average burst size, in reasonable agreement with the hypothesis of an enhanced copy error probability per round of geometrical DNA replication, the enhanced misreplication being caused by pyrimidine dimers in host DNA. The frequency distribution of c mutants produced from irradiated λc+ infecting irradiated host bacteria was fairly uniform when grouped into clones of size 2n (n = 0, 1, 2, …), over the mutant clone size-range from as large as complete bursts to clones of size two, but not for clones of size one. The uniformly distributed mutant clone sizes are explicable by a misrepair model in which the damage-bearing genes possess a high probability of mutating during one of the successive replication acts if, and only if, the damage is exposed, by chance, to an error-prone postreplication repair process induced by ultraviolet light in the host bacterium. Indirect and direct mutageneses in λ induced by ultraviolet radication were dissimilar in dependence on recA+, lex+, polA+ and certain other genes relevant to DNA repair. The results support the notion that there are multiple pathways for ultraviolet radiation-induced mutagenesis. Yields of indirectly induced mutants did not increase with increasing burst sizes, as they should if λ DNA replication is geometrical, supporting the model that initial geometrical replication is switched later to linear replication of the rollingcircle type.

Persistence and decay of thermoinducible error-prone repair activity in nonfilamentous derivatives of tif-1, Escherichia coli B/r: the timing of some critical events in ultraviolet mutagenesis.

Ultraviolet (UV) mutagenesis in E. coli is associated with a UV-inducible type of error-prone postreplication repair ("SOS" repair) which, in tif-1 strains, is thermo-inducible in coordination with other recA+ lexA+-dependent inducible functions, including filamentous growth. Mutants of E. coli B/r tif-1 strains have been isolated which retain thermoinducibility of SOS repair activity, but lack the thermosensitivity caused by filamentous growth at 42 degrees C. These strains have been used to determine: the kinetics of decay at 30 degrees C of thermally induced ability to enhance UV mutagenesis; the kinetics of thermal enhancement of spontaneous and UV-induced mutability at 42 degrees C, and the kinetics of decay at 30 degrees C of susceptibility to thermal enhancement of spontaneous and UV-induced mutability. Mutations from tryptophane requirement to prototrophy (Trp- to Trp+) were scored. UV doses were 0.2 J/M2 for excision repair-deficient (Uvr-) and 2J/m2 for Uvr+ strains. The results support the following conclusions. 1) thermally induced SOS repair activity decays at 30 degrees C to about 25% of its maximum level in 45 min, and is no longer detectable after 90 min. 2) Thermal enhancement of UV mutability occurs at sites produced primarily (perhaps exclusively) before completion of the first post-irradiation cell division. 3) UV-induced sites susceptible to thermally induced SOS repair are stable at 30 degrees C in cells not containing the error-prone repair system, and are refractory to constitutive error-free repair for at least 2-3 hours. 4) UV produces a potentially mutagenic type of photoproduct in DNA which can, without interacting with another UV lesion, provide a site susceptible to SOS repair, but which is not a sufficient signal for SOS induction. 5) 50-70% of the SOS-mutable SOS-noninducing UV photoproducts are photoreversible pyrimidine dimers. The results are discussed in relation to current models of UV mutagenesis and induction of UV-inducible functions.