Polyubiquitinated PCNA Recruits the ZRANB3 Translocase to Maintain Genomic Integrity after Replication Stress

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in?vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA?structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.     

RNA determinants of junction site selection in RNA virus recombinants and defective interfering RNAs.

RNA recombination plays an important role in the diversification and evolution of RNA viruses. Most of these events are believed to be mediated by an actively copying viral replicase switching from a donor template to an acceptor template, where it resumes synthesis. In addition, intramolecular replicase-mediated events (i.e., rearrangements) can lead to the generation of replicable deleted forms of a viral genome, termed defective interfering (DI) RNAs. To gain further insight into the recombination process, the effect of various primary and secondary structures on recombination site selection in vivo was examined using plant RNA tombusviruses. The effect of sequence identity and complementarity on deletion events that generate DI RNAs was also investigated. Our results suggest that (1) 5' termini and strong hairpin structures in donor templates represent preferred sites for recombinations, (2) junction sites in acceptor templates do not occur in double-stranded regions, (3) nucleotide homology can shift donor and acceptor recombination sites closer to regions of identity and, (4) both sequence identity and complementarity can direct deletion sites in DI RNAs. These results further define RNA determinants of tombusvirus RNA recombination and rearrangement.     

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.     

Human immunodeficiency virus type 1 genetic recombination is more frequent than that of Moloney murine leukemia virus despite similar template switching rates

Retroviral recombinants result from template switching between copackaged viral genomes. Here, marker reassortment between coexpressed vectors was measured during single replication cycles, and human immunodeficiency virus type 1 (HIV-1) recombination was observed six- to sevenfold more frequently than murine leukemia virus (MLV) recombination. Template switching was also assayed by using transduction-type vectors in which donor and acceptor template regions were joined covalently. In this situation, where RNA copackaging could not vary, MLV and HIV-1 template switching rates were indistinguishable. These findings argue that MLV's lower intermolecular recombination frequency does not reflect enzymological differences. Instead, these data suggest that recombination rates differ because coexpressed MLV RNAs are less accessible to the recombination machinery than are coexpressed HIV RNAs. This hypothesis provides a plausible explanation for why most gammaretrovirus recombinants, although relatively rare, display evidence of multiple nonselected crossovers. By implying that recombinogenic template switching occurs roughly four times on average during the synthesis of every MLV or HIV-1 DNA, these results suggest that virtually all products of retroviral replication are biochemical recombinants.     

Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability

Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.     

Mechanism of RNA recombination in carmo- and tombusviruses: evidence for template switching by the RNA-dependent RNA polymerase in vitro

RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligation-based RNA recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.     

Reduced dNTP interaction of human immunodeficiency virus type 1 reverse transcriptase promotes strand transfer

We have recently demonstrated that HIV-1 RT mutants characterized by low dNTP binding affinity display significantly reduced dNTP incorporation kinetics in comparison to wild-type RT. This defect is particularly emphasized at low dNTP concentrations where WT RT remains capable of efficient synthesis. Kinetic interference in DNA synthesis can induce RT pausing and slow down the synthesis rate. RT stalling and slow synthesis rate can enhance RNA template cleavage by RT-RNase H, facilitating transfer of the primer to a homologous template. We therefore hypothesized that reduced dNTP binding RT mutants can promote template switching during minus strand synthesis more efficiently than WT HIV-1 RT at low dNTP concentrations. To test this hypothesis, we employed two dNTP binding HIV-1 RT mutants, Q151N and V148I. Indeed, as the dNTP concentration was decreased, the template switching frequency progressively increased for both WT and mutant RTs. However, as predicted, the RT mutants promoted more transfers compared with WT RT. The WT and mutant RTs were similar in their intrinsic RNase H activity, supporting that the elevated template switching efficiency of the mutants was not the result of the mutations enhancing RNase H activity. Rather, kinetic interference leading to stalled DNA synthesis likely enhanced transfers. These results suggest that the RT-dNTP substrate interaction mechanistically influences strand transfer and recombination of HIV-1 RT.     

The FANCM family of DNA helicases/translocases

FANCM and its relatives, Hef, Mph1 and Fml1, are DNA junction-specific helicases/translocases that target and process perturbed replication forks and intermediates of homologous recombination. They have variously been implicated in promoting the activation of the S-phase checkpoint, recruitment of the Fanconi Anemia Core Complex to sites of DNA damage, crossover avoidance during DNA double-strand break repair by homologous recombination, and the replicative bypass of DNA lesions by template switching. This review summarises our current understanding of the biochemical activities and biological functions of the FANCM family.     

DNA bending facilitates the error‐free DNA damage tolerance pathway and upholds genome integrity

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination‐mediated (error‐free) and translesion synthesis‐mediated (error‐prone) DNA damage tolerance pathways. Crucial for error‐free DNA damage tolerance is template switching, which depends on the formation and resolution of damage‐bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication‐associated lesions into the error‐free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C‐terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication‐associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.     

Initiation of genetic recombination and recombination-dependent replication

Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.     

RNA-guided DNA assembly

We propose molecular models for homologous DNA recombination events that are guided by either double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) templates. The models are applied to explain DNA rearrangements in some groups of ciliates, such as Stylonychia or Oxytricha, where extensive gene rearrangement occurs during differentiation of a somatic macronucleus from a germline micronucleus. We describe a model for RNA template guided DNA recombination, such that the template serves as a catalyst that remains unchanged after DNA recombination. This recombination can be seen as topological braiding of the DNA, with the template-guided alignment proceeding through DNA branch migration. We show that a virtual knot diagram can provide a physical representation of the DNA at the time of recombination. Schematically, the braiding process can be represented as a crossing in the virtual knot diagram. The homologous recombination corresponds to removal of the crossings in the knot diagram (called smoothing). We show that if all recombinations are performed at the same time (i.e., simultaneous smoothings of the crossings) then one of the resulting DNA molecules will always contain all of the gene segments in their correct, linear order, which produces the mature DNA sequence.