Human RAD52 stimulates the RAD51-mediated homology search.

Homologous recombination (HR) is a DNA repair mechanism of double-strand breaks and blocked replication forks, involving a process of homology search leading to the formation of synaptic intermediates that are regulated to ensure genome integrity. RAD51 recombinase plays a central role in this mechanism, supported by its RAD52 and BRCA2 partners. If the mediator function of BRCA2 to load RAD51 on RPA-ssDNA is well established, the role of RAD52 in HR is still far from understood. We used transmission electron microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52, and BRCA2 in the assembly of the RAD51 filament and its activity. Although our results confirm that RAD52 lacks a mediator activity, RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator activity of BRCA2, and form shorter RAD51-RAD52 mixed filaments that are more efficient in the formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. We confirm the in situ interaction between RAD51 and RAD52 after double-strand break induction in vivo. This study provides new molecular insights into the formation and regulation of presynaptic and synaptic intermediates by BRCA2 and RAD52 during human HR.

Changes in the architecture and abundance of replication intermediates delineate the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells.

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

SUMO-mediated recruitment allows timely function of the Yen1 nuclease in mitotic cells.

The post-translational modification of DNA damage response proteins with SUMO is an important mechanism to orchestrate a timely and orderly recruitment of repair factors to damage sites. After DNA replication stress and double-strand break formation, a number of repair factors are SUMOylated and interact with other SUMOylated factors, including the Yen1 nuclease. Yen1 plays a critical role in ensuring genome stability and unperturbed chromosome segregation by removing covalently linked DNA intermediates between sister chromatids that are formed by homologous recombination. Here we show how this important role of Yen1 depends on interactions mediated by non-covalent binding to SUMOylated partners. Mutations in the motifs that allow SUMO-mediated recruitment of Yen1 impair its ability to resolve DNA intermediates and result in chromosome mis-segregation and increased genome instability.

The FANCM family Mph1 helicase localizes to the mitochondria and contributes to mtDNA stability.

Mitochondria are membrane-bound organelles found in eukaryotic cells where they generate energy through the respiratory chain. They contain their own genome that encodes genes critical to the mitochondrial function, but most of their protein content is synthetized from nuclear encoded genes. Damages to the mtDNA can cause mutations and rearrangements with an impact on the respiratory functions of the cell. DNA repair factors are able to localize to mitochondria to restore mtDNA integrity and ensure its proper inheritance. We describe in this article the mitochondrial localization of the Mph1/FANCM helicase that serves critical roles in nuclear DNA repair processes. Mph1 localizes to mitochondria and its functions contribute to the mtDNA integrity under mtDNA damaging conditions.

Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli.

BACKGROUND: DNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2'-deoxyguanosine-5'-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. METHODS: Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproduct (6-4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured. RESULTS: We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6-4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains. DISCUSSION: These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

Slx5-Slx8 ubiquitin ligase targets active pools of the Yen1 nuclease to limit crossover formation.

The repair of double-stranded DNA breaks (DSBs) by homologous recombination involves the formation of branched intermediates that can lead to crossovers following nucleolytic resolution. The nucleases Mus81-Mms4 and Yen1 are tightly controlled during the cell cycle to limit the extent of crossover formation and preserve genome integrity. Here we show that Yen1 is further regulated by sumoylation and ubiquitination. In vivo, Yen1 becomes sumoylated under conditions of DNA damage by the redundant activities of Siz1 and Siz2 SUMO ligases. Yen1 is also a substrate of the Slx5-Slx8 ubiquitin ligase. Loss of Slx5-Slx8 stabilizes the sumoylated fraction, attenuates Yen1 degradation at the G1/S transition, and results in persistent localization of Yen1 in nuclear foci. Slx5-Slx8-dependent ubiquitination of Yen1 occurs mainly at K714 and mutation of this lysine increases crossover formation during DSB repair and suppresses chromosome segregation defects in a mus81∆ background.

The nucleolytic resolution of recombination intermediates in yeast mitotic cells.

In mitotic cells, the repair of double-strand breaks by homologous recombination (HR) is important for genome integrity. HR requires the orchestration of a subset of pathways for timely removal of joint-molecule intermediates that would otherwise prevent segregation of chromosomes in mitosis. The use of nucleases to resolve recombination intermediates is important for chromosome segregation, but is hazardous because crossovers can result in loss of heterozygosity or chromosome rearrangements. Unregulated use of the nucleases involved in the resolution of recombination intermediates could also be a risk during replication. The yeast models (Saccharomyces cerevisae and Schizosaccharomyces pombe) have proven effective in determining the major nucleases involved in the processing of such intermediates: Mus81-Mms4 and Yen1. Mus81-Mms4 and Yen1 are regulated by the cell cycle in a gradual activation during G2/M to keep the crossing-over risk low while ensuring proper removal of HJ intermediates.

Ethylene oxide and propylene oxide derived N7-alkylguanine adducts are bypassed accurately in vivo.

Adducts formed at the nucleophilic N7 position of guanine are the most abundant lesions produced by alkylating agents such as ethylene oxide (EO) and propylene oxide (PO). In order to investigate the intrinsic mutagenic potential of N7-alkylguanine adducts, we prepared single-stranded DNA probes containing a single well-defined N7-alkylguanine adduct under conditions that minimize the presence of depurinated molecules. Following introduction of these probes into Escherichia coli cells, the effect of the N7-alkylguanine adducts on the efficiency and fidelity of replication was determined. To investigate the effect on replication we monitored the relative transformation efficiency of the lesion containing constructs with respect to the control construct. The methyl adduct was found not to be toxic, while the N7-(2-hydroxyethyl)guanine (N7-heG) and N7-(2-hydroxypropyl)guanine (N7-hpG) adducts reduce the transformation efficiency to ≈70% and 40%, respectively. Within the detection limits of our assay, replication across the N7-alkylguanine adducts in vivo is essentially error-free, as no mutant colony was observed among ≈300 individual sequenced colonies (i.e., mutation frequency<0.3%).

The Cdk/cDc14 module controls activation of the Yen1 holliday junction resolvase to promote genome stability.

Faithful genome transmission during cell division requires precise, coordinated action of DNA metabolic enzymes, including proteins responsible for DNA damage detection and repair. Dynamic phosphorylation plays an important role in controlling repair enzymes during the DNA damage response (DDR). Cdc14 phosphatases oppose cyclin-dependent kinase (Cdk) phosphorylation and have been implicated in the DDR in several model systems. Here, we have refined the substrate specificity of budding yeast Cdc14 and, using this insight, identified the Holliday junction resolvase Yen1 as a DNA repair target of Cdc14. Cdc14 activation at anaphase triggers nuclear accumulation and enzymatic activation of Yen1, likely to resolve persistent recombinational repair intermediates. Consistent with this, expression of a phosphomimetic Yen1 mutant increased sister chromatid nondisjunction. In contrast, lack of Cdk phosphorylation resulted in constitutive activity and elevated crossover-associated repair. The precise timing of Yen1 activation, governed by core cell-cycle regulators, helps coordinate DNA repair with chromosome segregation and safeguards against genome destabilization.

Mph1 and Mus81-Mms4 prevent aberrant processing of mitotic recombination intermediates.

Homology-dependent repair of double-strand breaks (DSBs) from nonsister templates has the potential to generate loss of heterozygosity or genome rearrangements. Here we show that the Saccharomyces cerevisiae Mph1 helicase prevents crossovers between ectopic sequences by removing substrates for Mus81-Mms4 or Rad1-Rad10 cleavage. A role for Yen1 is only apparent in the absence of Mus81. Cells lacking Mph1 and the three nucleases are highly defective in the repair of a single DSB, suggesting that the recombination intermediates that accumulate cannot be processed by the Sgs1-Top3-Rmi1 complex (STR). Consistent with this hypothesis, ectopic joint molecules (JMs) accumulate transiently in the mph1Δ mutant and persistently when Mus81 is eliminated. Furthermore, the ectopic JMs formed in the mus81Δ mutant contain a single Holliday junction (HJ) explaining why STR is unable to process them. We suggest that Mph1 and Mus81-Mms4 recognize an early strand exchange intermediate and direct repair to noncrossover or crossover outcomes, respectively.

The Rad1-Rad10 nuclease promotes chromosome translocations between dispersed repeats.

Holliday junctions can be formed during homology-dependent repair of DNA double-strand breaks, and their resolution is essential for chromosome segregation and generation of crossover products. The Mus81-Mms4 and Yen1 nucleases are required for mitotic crossovers between chromosome homologs in Saccharomyces cerevisiae; however, crossovers between dispersed repeats are still detected in their absence. Here we show that the Rad1-Rad10 nuclease promotes formation of crossover and noncrossover recombinants between ectopic sequences. Crossover products were not recovered from the mus81Δ rad1Δ yen1Δ triple mutant, indicating that all three nucleases participate in processing recombination intermediates that form between dispersed repeats. We suggest a new mechanism for crossovers that involves Rad1-Rad10 clipping and resolution of a single Holliday junction-containing intermediate by Mus81-Mms4 or Yen1 cleavage or by replication. Consistent with the model, we show accumulation of Rad1-dependent joint molecules in the mus81Δ yen1Δ mutant.

Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome.

Although most deoxyribonucleic acid (DNA) lesions are accurately repaired before replication, replication across unrepaired lesions is the main source of point mutations. The lesion tolerance processes, which allow damaged DNA to be replicated, entail two branches, error-prone translesion synthesis (TLS) and error-free damage avoidance (DA). While TLS pathways are reasonably well established, DA pathways are poorly understood. The fate of a replication-blocking lesion is generally explored by means of plasmid-based assays. Although such assays represent efficient tools to analyse TLS, we show here that plasmid-borne lesions are inappropriate models to study DA pathways due to extensive replication fork uncoupling. This observation prompted us to develop a method to graft, site-specifically, a single lesion in the genome of a living cell. With this novel assay, we show that in Escherichia coli DA events massively outweigh TLS events and that in contrast to plasmid, chromosome-borne lesions partially require RecA for tolerance.

Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast.

Holliday junction (HJ) resolution is required for segregation of chromosomes and for formation of crossovers during homologous recombination. The identity of the resolvase(s) that functions in vivo has yet to be established, although several proteins able to cut HJs in vitro have been identified as candidates in yeasts and mammals. Using an assay to detect unselected products of mitotic recombination, we found a significant decrease in crossovers in the Saccharomyces cerevisiae mus81Δ mutant. Yen1 serves a backup function responsible for resolving intermediates in mus81Δ mutants, or when conversion tracts are short. In the absence of both Mus81 and Yen1, intermediates are not channeled exclusively to noncrossover recombinants, but instead are processed by Pol32-dependent break-induced replication (BIR). The channeling of recombination from reciprocal exchange to BIR results in greatly increased spontaneous loss of heterozygosity (LOH) and chromosome mis-segregation in the mus81Δ yen1Δ mutant, typical of the genomic instability found in tumor cells.

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O6-alkylguanine adducts in Escherichia coli.

O(6)-alkylG adducts are highly mutagenic due to their capacity to efficiently form O(6)-alkylG:T mispairs during replication, thus triggering G→A transitions. Mutagenesis is largely prevented by repair strategies such as reversal by alkyltransferases or excision by nucleotide excision repair (NER). Moreover, methyl-directed mismatch repair (MMR) is known to trigger sensitivity to methylating agents via a mechanism that involves recognition by MutS of the O(6)-mG:T replication intermediates. We wanted to investigate the mechanism by which MMR controls the genotoxicity of environmentally relevant O(6)-alkylG adducts formed by ethylene oxide and propylene oxide. Recently, the alkyltransferase-like gene ybaZ (eATL) was shown to enhance repair of these slightly larger O(6)-alkylG adducts by NER. We analyzed the toxicity and mutagenesis induced by these O(6)-alkylG adducts using single-adducted plasmid probes. We show that the eATL gene product prevents MMR-mediated attack of the O(6)-alkylG:T replication intermediate for the larger alkyl groups but not for methyl. In vivo data are compatible with the occurrence of repeated cycles of MMR attack of the O(6)-alkylG:T intermediate. In addition, in vitro, the eATL protein efficiently prevents binding of MutS to the O(6)-alkylG:T mispairs formed by the larger alkyl groups but not by methyl. In conclusion, eATL not only enhances the efficiency of repair of these larger adducts by NER, it also shields these adducts from MMR-mediated toxicity.

The alkyltransferase-like ybaZ gene product enhances nucleotide excision repair of O(6)-alkylguanine adducts in E. coli.

O(6)-methylguanine adducts are potent pre-mutagenic lesions owing to their high capacity to direct mis-insertion of thymine when bypassed by replicative DNA polymerases. The strong mutagenic potential of these adducts is prevented by alkyltransferases such as Ada and Ogt in Escherichia coli that transfer the methyl group to one of their cysteine residues. Alkyl residues larger than methyl are generally weak substrates for reversion by alkyltransferases. In this paper we have investigated the genotoxic potential of the O(6)-alkylguanine adducts formed by ethylene and propylene oxide using single-adducted plasmid probes. Our work shows that the ybaZ gene product, a member of the alkyltransferase-like protein family, strongly enhances the repair by nucleotide excision repair of the larger O(6)-alkylguanine adducts that are otherwise poor substrates for alkyltransferases. The YbaZ protein is shown to interact with UvrA. This factor may thus enhance the efficiency of nucleotide excision repair in a way similar to the Transcription-Repair Coupling factor Mfd, by recruiting the UvrA(2).UvrB complex to the adduct site via its interaction with UvrA.

Insights into the LexA regulon of Thermotogales.

The lexA genes of Thermotoga maritima and Petrotoga miotherma, both members of the Order Thermotogales, have been cloned and their transcriptional organization, as well as the functional characteristics of their encoded products, analyzed. In both bacterial species, the lexA gene was found to be co-transcribed together with another four (T. maritima) or three (P. miotherma) upstream open-reading frames. The P. miotherma LexA was able to bind promoters of both the cognate lexA encoding operon and the uvrA gene but not to that of the recA. Conversely, LexA protein and crude cell extracts from T. maritima were unable to bind promoters governing the expression of either its lexA or recA genes. In agreement with these observations, no functional copy of the P. miotherma LexA box, corresponding to the GANTN(6)GANNAC motif, seems to be present in the T. maritima genome. Giving support to the proposal that the evolutionary branching order of the Order Thermotogales is very close to that of Gram-positive bacteria, the P. miotherma LexA protein was still able to recognize the previously described LexA-binding sequence for Gram-positive bacteria.

Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria lexA gene.

Acidobacterium capsulatum is the most thoroughly studied species of a new bacterial phylogenetic group designated the phylum Acidobacteria. Through a tblastn search, the A. capsulatum lexA gene has been identified, and its product purified. Electrophoretic mobility shift assays have shown that A. capsulatum LexA protein binds specifically to the direct repeat GTTCN(7)GTTC motif. Strikingly, this is also the LexA box of the Alphaproteobacteria, but had not previously been described outside this subclass of the Proteobacteria. In addition, a phylogenetic analysis of the LexA protein clusters together Acidobacterium and the Alphaproteobacteria, moving the latter away from their established phylogenetic position as a subclass of the Proteobacteria, and pointing to a lateral gene transfer of the lexA gene from the phylum Acidobacteria, or an immediate ancestor, to the Alphaproteobacteria. Lastly, in vivo experiments demonstrate that the A. capsulatum recA gene is DNA-damage inducible, despite the fact that a LexA-binding sequence is not present in its promoter region.

Dispersal and regulation of an adaptive mutagenesis cassette in the bacteria domain.

Recently, a multiple gene cassette with mutagenic translation synthesis activity was identified and shown to be under LexA regulation in several proteobacteria species. In this work, we have traced down instances of this multiple gene cassette across the bacteria domain. Phylogenetic analyses show that this cassette has undergone several reorganizations since its inception in the actinobacteria, and that it has dispersed across the bacterial domain through a combination of vertical inheritance, lateral gene transfer and duplication. In addition, our analyses show that LexA regulation of this multiple gene cassette is persistent in all the phyla in which it has been detected, and suggest that this regulation is prompted by the combined activity of two of its constituent genes: a polymerase V homolog and an alpha subunit of the DNA polymerase III.

The Leptospira interrogans lexA gene is not autoregulated.

Footprinting and mutagenesis experiments demonstrated that Leptospira interrogans LexA binds the palindrome TTTGN(5)CAAA found in the recA promoter but not in the lexA promoter. In silico analysis revealed that none of the other canonical SOS genes is under direct control of LexA, making the leptospiral lexA gene the first described which is not autoregulated.