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Mechanisms of recombination between diverged sequences in wild-type and BLM-deficient mouse and human cells.

ABSTRACT: Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged ("homeologous") sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells and are generally < or =100 bp, even in Msh2(-)(/)(-) cells which fail to suppress homeologous recombination. Gene conversion tracts are mostly unidirectional, with no observed mutations. Additionally, no alterations were observed in the donor sequences. While both mouse and human cells suppress homeologous recombination, the suppression is substantially less in the transformed human cells, despite similarities in the gene conversion tracts. BLM-deficient mouse and human cells suppress homeologous recombination to a similar extent as wild-type cells, unlike Sgs1-deficient Saccharomyces cerevisiae.

SUBMITTER: Larocque JR

PROVIDER: S-EPMC2849462 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Mechanisms of recombination between diverged sequences in wild-type and BLM-deficient mouse and human cells.

Larocque Jeannine R JR Jasin Maria M

Molecular and cellular biology 20100212 8

Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recom ...[more]

PMID: 20154148

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Project description:V(D)J recombination is a highly regulated process, proceeding from a site-specific cleavage to an imprecise end joining. After the DNA excision catalyzed by the recombinase encoded by recombination activating genes 1 and 2 (RAG1/2), newly generated recombination ends are believed held by a post-cleavage complex (PC) consisting of RAG1/2 proteins, and are subsequently resolved by non-homologous end joining (NHEJ) machinery. The relay of these ends from PC to NHEJ remains elusive. It has been speculated that NHEJ factors modify the RAG1/2-PC to gain access to the ends or act on free ends after the disassembly of the PC. Thus, recombination ends may either be retained in a complex throughout the recombination process or left as unprotected free ends after cleavage, a condition that may permit an alternative, non-classical NHEJ end joining pathway. To directly test these scenarios on recombination induced chromosomal breaks, we have developed a recombination end protection assay to monitor the accessibility of recombination ends to exonuclease-V in intact nuclei. We demonstrate that these ends are well protected in the nuclei of wild-type cells, suggesting a seamless cleavage-joining reaction. However, divergent end protection of coding versus signal ends was found in cells derived from severe combined immunodeficient (scid) mice that are defective in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While signal ends are resistant, opened coding ends are susceptible to enzymatic modification. Our data suggests a role of DNA-PKcs in protecting chromosomal coding ends. Furthermore, using recombination inducible scid cell lines, we demonstrate that conditional protection of coding ends is inversely correlated with the level of their resolution, i.e., the greater the accessibility of the coding ends, the higher level of coding joints formed. Taken together, our findings provide important insights into the resolution of recombination ends by error-prone alternative NHEJ pathways.

Dataset Information

Mechanisms of recombination between diverged sequences in wild-type and BLM-deficient mouse and human cells.

Publications

Mechanisms of recombination between diverged sequences in wild-type and BLM-deficient mouse and human cells.

Similar Datasets

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