Regulation of Homologous Recombination between Divergent DNA Sequences by Mismatch Repair Proteins

Abstract

Genomes of living organisms, from unicellular bacteria to multicellular human, are threatened by a plethora of DNA lesions. It is estimated that there are ~100,000 DNA lesions inflicted in a single cell on a daily basis (Lindahl, 1993). Both endogenous and exogenous agents induce the formation of these lesions in the genome. Internally, other than replication errors that generate DNA mismatches, reactive oxygen species, alkylating agents and spontaneous hydrolysis damage DNA. These structural alterations include oxidation, methylation, deamination, depurination and depyrimidation of DNA bases. External agents such as ultraviolet radiation, high-frequency radiations like X-rays and γ-rays, natural and synthetic toxins, can all damage DNA by changing its structure. These agents mediate the formation of intra- and interstrand crosslinks, of bulky adducts as well as of single- and double-strand breaks (DSBs) in the DNA. To ensure proper operation of DNA transactions, which are important for cellular survival, a variety of DNA-repair pathways act on these DNA lesions to preserve the integrity of the genome (Hoeijmakers 2001, 2009; Friedberg 2003; Garinis et al., 2008). These DNArepair pathways include DNA mismatch repair (MMR; Jiricny 2013), base excision repair (BER; Goosen and Moolenaar, 2008), nucleotide excision repair (NER; Goosen and Moolenaar, 2008), non-homologous end joining (NHEJ; Shuman and Glickman, 2007) and homologous recombination (HR; Kowalczykowski et al., 1994-1; Heyer et al., 2010). Among the various types of DNA lesions, DSBs are particularly toxic. The failure to correctly process DSBs result in genome instability and has been associated with cancer predisposition, immune deficiency and infertility (Jackson and Bartek, 2009; McKinnon, 2009). Two prominent pathways that repair DSB are the error-prone NHEJ and the largely error-free HR, which uses the additional copies of homologous DNA sequences, present during the S and G2 phases of the cell cycle in eukaryotes, as repair template (Takata et al., 1998). However, DNA sequences of sister chromatids and homologous chromosomes are not always identical. Recombination between such DNA partners (hereafter referred to as homeologous recombination) generates mismatches within the heteroduplex region of the strand-exchange products. Similarly, recombination between repetitive DNA in eukaryotic genomes generates mismatches as well (George and Alani, 2012). These mismatches in turn act as DNA substrates for the activation of the MMR pathway. Depending on which strand within the heteroduplex is targeted by MMR proteins, mismatches result in gene conversion or simply restoration (Surtees et al., 2004). This could occur either during strand exchange or after the formation of crossover and non-crossover recombination products. MMR proteins additionally modulate HR to prevent recombination between divergent sequences. How the Escherichia coli MMR proteins impose homeologous antirecombination is the main question addressed in this thesis.