Assessment of Anti-recombination and Double-strand Break-induced Gene Conversion in Human Cells by a Chromosomal Reporter

Abstract

Gene conversion is one of the frequent end results of homologous recombination, and it often underlies the inactivation of tumor suppressor genes in cancer cells. Here, we have developed an integrated assay system that allows simultaneous examination of double-strand break (DSB)-induced gene conversion events at the site of a DSB (proximal region) and at a surrounding region ∼1 kb away from the break (distal region). Utilizing this assay system, we find that gene conversion events at the proximal and distal regions are relatively independent of one another. The results also indicate that synthesis-dependent strand annealing (SDSA) plays a major role in DSB-induced gene conversion. In addition, our current study has demonstrated that hMLH1 plays an essential role in anti-recombination and gene conversion. Specifically, the anti-recombination activity of hMLH1 is partially dependent on its interaction with hMRE11. Our data suggests that the role of hMLH1 and hMRE11 in the process of gene conversion is complex, and these proteins play different roles in DSB-induced proximal and distal gene conversions. In particular, the involvement of hMLH1 and hMRE11 in the distal gene conversion requires both hMSH2 and heteroduplex formation. Gene conversion is one of the frequent end results of homologous recombination, and it often underlies the inactivation of tumor suppressor genes in cancer cells. Here, we have developed an integrated assay system that allows simultaneous examination of double-strand break (DSB)-induced gene conversion events at the site of a DSB (proximal region) and at a surrounding region ∼1 kb away from the break (distal region). Utilizing this assay system, we find that gene conversion events at the proximal and distal regions are relatively independent of one another. The results also indicate that synthesis-dependent strand annealing (SDSA) plays a major role in DSB-induced gene conversion. In addition, our current study has demonstrated that hMLH1 plays an essential role in anti-recombination and gene conversion. Specifically, the anti-recombination activity of hMLH1 is partially dependent on its interaction with hMRE11. Our data suggests that the role of hMLH1 and hMRE11 in the process of gene conversion is complex, and these proteins play different roles in DSB-induced proximal and distal gene conversions. In particular, the involvement of hMLH1 and hMRE11 in the distal gene conversion requires both hMSH2 and heteroduplex formation.