Academic journal article Genetics

A Role for DNA Mismatch Repair Protein Msh2 in Error-Prone Double-Strand-Break Repair in Mammalian Chromosomes

Academic journal article Genetics

A Role for DNA Mismatch Repair Protein Msh2 in Error-Prone Double-Strand-Break Repair in Mammalian Chromosomes

Article excerpt

ABSTRACT

We examined error-prone nonhomologous end joining (NHEJ) in Msh2-deficient and wild-type Chinese hamster ovary cell lines. A DNA substrate containing a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene was stably integrated into cells. The fusion gene was rendered nonfunctional due to a 22-bp oligonucleotide insertion, which included the 18-bp I-SceI endonuclease recognition site, within the tk portion of the fusion gene. A double-strand break (DSB) was induced by transiently expressing the I-SceI endonuclease, and deletions or insertions that restored the tk-neo fusion gene's reading frame were recovered by selecting for G418-resistant colonies. Overall, neither the frequency of recovery of G418-resistant colonies nor the sizes of NHEJ-associated deletions were substantially different for the mutant vs. wild-type cell lines. However, we did observe greater usage of terminal microhomology among NHEJ events recovered from wild-type cells as compared to Msh2 mutants. Our results suggest that Msh2 influences error-prone NHEJ repair at the step of pairing of terminal DNA tails. We also report the recovery from both wild-type and Msh2-deficient cells of an unusual class of NHEJ events associated with multiple deletion intervals, and we discuss a possible mechanism for the generation of these "discontinuous deletions."

MAMMALIAN cells contend with various forms of DNA damage on a daily basis. One type of DNA damage that is potentially quite deleterious is a doublestrand break (DSB). DSBs can arise at stalled replication forks or following exposure to a variety of chemical or radiological agents. At least two general pathways for DSB repair in eukaryotes exist: homologous recombination, and nonhomologous end joining (NHEJ) (Gnu 1997; LIANG et al 1998; LIN et al 1999; HABER 2000; KARRAN 2000; FERGUSON and ALT 2001; JOHNSON and JASIN 2001; KHANNA and JACKSON 2001; NORBURY and HICKSON 2001; PASTINK et al 2001; PIERCE et al 2001; VAN GENT et al. 2001; BERNSTEIN et al. 2002; JACKSON 2002; HELLEDAY 2003; VALERIE and POVIRK 2003). Homologous recombination is an accurate repair pathway utilizing a homologous DNA template to correctly restore genetic information that may otherwise be lost at a DSB site. In contrast, NHEJ involves no template and is error prone because one or several nucleotides are usually deleted or inserted prior to DSB healing. NHEJ is considered to be a major DSB repair pathway in mammalian cells.

One approach toward gaining a better understanding of DSB repair pathways is to study the consequences of the loss of specific proteins that appear to be reasonable candidates for involvement in repair. It has been observed that NHEJ repair junctions often occur within short patches of homology, suggesting that the joining of DNA ends via NHEJ may be facilitated by terminal microhomologies (Gnu 1997; LIANG et al. 1998; LIN et al 1999; HABER 2000; KARRAN 2000; FERGUSON and ALT 2001; JOHNSON and JASIN 2001; KHANNA and JACKSON 2001; NORBURY and HICKSON 2001; PASTINK et al 2001; PIERCE et al. 2001; VAN GENT et al. 2001; BERNSTEIN et al. 2002; JACKSON 2002; HELLEDAY 2003; VALERIE and POVIRK 2003). One may envision that NHEJ involves interactions between single-stranded DNA tails with a concomitant "search" for homology as the strands align for subsequent joining. The homology search may be directed by specific proteins or may be driven by spontaneous base pairing. Interactions between mismatched DNA tails may produce short segments of heteroduplex DNA containing mispaired bases, and these mispairs may be substrates for the DNA mismatch repair (MMR) machinery. For this, and other reasons elaborated below, it seems reasonable to think that MMR proteins have a role in NHEJ.

In eukaryotes, Msh2 (MutS homologue 2) is a major player in MMR, functioning as a heterodimer in association with Msh3 or Msh6. Msh2 has been implicated in a variety of processes that serve to protect genomic integrity. …

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