Academic journal article Genetics

Microhomology-Mediated End Joining in Fission Yeast Is Repressed by Pku70 and Relies on Genes Involved in Homologous Recombination

Academic journal article Genetics

Microhomology-Mediated End Joining in Fission Yeast Is Repressed by Pku70 and Relies on Genes Involved in Homologous Recombination

Article excerpt

ABSTRACT

Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joining (MMEJ) mechanism in fission yeast. MMEJ was found to require at least five homologous nucleotides and its efficiency was decreased by the presence of nonhomologous nucleotides either within the overlapping sequences or at DSB ends. Exo1 exonuclease and Rad22, a Rad52 homolog, were required for repair, suggesting that MMEJ is related to the single-strand-annealing (SSA) pathway of HR. In addition, MMEJ-dependent repair of DSBs with discontinuous microhomologies was strictly dependent on Pol4, a PolX DNA polymerase. Although not strictly required, Msh2 and Pms1 mismatch repair proteins affected the pattern of MMEJ repair. Strikingly, Pku70 inhibited MMEJ and increased the minimal homology length required for efficient MMEJ. Overall, this study strongly suggests that MMEJ does not define a distinct DSB repair mechanism but reflects "micro-SSA."

ONE of the most toxic lesions to DNA is the doublestrand break (DSB). If left unrepaired, DSB has the potential to disrupt genomic integrity. Notably, many cancers of lymphoid origin are due to defective DSB repair of V(D)J recombination intermediates (Jackson 2002). Cells have evolved two main pathways to repair DSBs: nonhomologous end joining (NHEJ), a process resulting in direct resealing of the break without the need of extended homology between both ends, and homologous recombination (HR), which, through at least 20bpofhomology(ShenandHuang1986), repairs breaks by copying genetic information from either homologous chromosomes or sister chromatids. There are at least three differentmechanisms ofHRinmitotic cells: gene conversion (GC), break-induced replication, and single-strand annealing (SSA); the latter mechanism repairs DSBs arising between direct repeats of homology, leading todeletionof theinterveningnucleotides (Paques and Haber 1999).

Genetic requirements for HR and NHEJ have been extensively investigated in a variety of organisms. In budding yeast, HR machinery includes genes from the RAD52 epistasis group [RAD52, RAD59, RAD51, RAD54, RAD55, and RAD57 (RAD51 family); RDH54 and RAD50, XRS2, and MRE11 (MRE11 family)] (Paques andHaber 1999). RAD52 encodes a single-standed DNA (ssDNA)-binding protein with single-strand annealing activity required for all HR events, including SSA, although the requirement for RAD52 in SSA diminishes as the length of homologous repeats flanking the DSB increases (.2 kb) (Ozenberger and Roeder 1991). Budding yeast RAD59, a RAD52 homolog with strand-annealing activity, is also required for SSA, especially when the homologous regions are short (Sugawara et al. 2000; Davis and Symington 2001). On the other hand, the ATPdependent strand exchange mediator Rad51, the homolog of bacterial RecA, is required for GC and the majority of break-induced replication events but not for SSA in budding yeast (Paques and Haber 1999; Davis and Symington 2004). The involvement of the Saccharomyces cerevisiae Mre11 complex (RAD50/XRS2/MRE11) in HR is still controversial although the DNA end-bridging activity ofRad50 could potentially stimulateHR (D'Amours and Jackson 2002). In Schizosaccharomyces pombe, it was suggested that Rad50 stimulates sister-chromatid recombination but not recombination between homologous chromosomes (Hartsuiker et al. 2001). Key proteins for NHEJ are the heterodimeric DNA-binding proteins Ku70/Ku80 andDNAligase IV (Jackson 2002). TheKu70/80 heterodimer is required for efficient and accurate NHEJ; it binds DNA ends and protects them from exonuclease digestion (Getts and Stamato 1994). …

Search by... Author
Show... All Results Primary Sources Peer-reviewed

Oops!

An unknown error has occurred. Please click the button below to reload the page. If the problem persists, please try again in a little while.