Interaction of Nick-directed DNA Mismatch Repair and Loop Repair in Human Cells

Abstract

In human cells, large DNA loop heterologies are repaired through a nick-directed pathway independent of mismatch repair. However, a 3-nick generated by bacteriophage fd gene II protein heterology is not capable of stimulating loop repair. To evaluate the possibility that a mismatch near a loop could induce both repair types in human cell extracts, we constructed and tested a set of DNA heteroduplexes, each of which contains a combination of mismatches and loops. We have demonstrated that a strand break generated by restriction endonucleases 3 to a large loop is capable of provoking and directing loop repair. The repair of 3-heteroduplexes in human cell extracts is very similar to that of 5-heteroduplex repair, being strand-specific and highly biased to the nicked strand. This observation suggests that the loop repair pathway possesses bidirectional repair capability similar to that of the bacterial loop repair system. We also found that a nick 5 to a coincident mismatch and loop can apparently stimulate the repair of both. In contrast, 3-nick-directed repair of a G-G mismatch was reduced when in the vicinity of a loop (33 or 46 bp between two sites). Increasing the distance separating the G-G mismatch and loop by 325 bp restored the efficiency of repair to the level of a single base-base mismatch. This observation suggests interference between 3-nick-directed large loop repair and conventional mismatch repair systems when a mispair is near a loop. We propose a model in which DNA repair systems avoid simultaneous repair at adjacent sites to avoid the creation of double-stranded DNA breaks.DNA loop heterologies can occur as a consequence of DNA biosynthetic errors or as a result of recombinational strand transfer between heterologous sequences (1-4). These DNA loop structures are mutagenic if they are not corrected. It is known that the DNA mismatch repair system in both prokaryotes and eukaryotes is capable of correcting small DNA loops (for reviews, see Refs. 5-10). The Escherichia coli mismatch repair system can efficiently process DNA loops up to 7 unpaired nucleotides (11). Genetic studies suggest that, in yeast, the mismatch repair system may be capable of correcting DNA loops up to 13 unpaired nucleotides (12). Functional in vitro mismatch repair assays have shown that the human mismatch repair pathway can repair DNA loops with at least 8 unpaired nucleotides (13).There is accumulating evidence suggesting that DNA loop repair can be carried out by a pathway distinct from the mismatch repair system (13-19). Conflicting results have been obtained regarding the strand specificity and the role of strand breaks in mammalian loop repair. Although single-stranded loops of varying sizes are repaired efficiently in vivo, there is a 2:1 bias against loop retention (20). Nicks have a minor effect on strand discrimination for biased repair of 25-57-base singlestranded loops in monkey cells (21). However, in vitro functional assays have been used to demonstrate that, in the absence of nicks, repair of multi-based loops ...

Intrachromosomal serial replication slippage intransgives rise to diverse genomic rearrangements involving inversions

“…Actually, SRS is predicted to result in a looped structure with unpaired bases on either the template or the nascent strand that is then subject to correction by the host repair system. While small loops that range from one to 16 nucleotides are corrected by mismatch repair, large DNA loops of up to several hundred nucleotides are repaired by a distinct process termed ''large loop repair'' [McCulloch et al, 2003;Corrette-Bennett et al, 2004;Huang et al, 2004]. However, in the case of SRS-derived mutations, it is the loop structure in the template strands that must be corrected.…”

Isolated short CTG/CAG DNA slip-outs are repaired efficiently by hMutSβ, but clustered slip-outs are poorly repaired

“…We suggest that closely spaced adjacent short slip-outs interfere with each other's repair, possibly serving as DNA structural blockades. Interference has been observed in the repair of adjacent G-T and U-G mispairs (38) and also in palindromic hairpins with adjacent G-T mispairs by human cell extracts (39)(40)(41). Notably, the degree of interference increased with diminished distance between the DNA lesions.…”

“…In bacteria, when two adjacent base-base mismatches are separated by 1 to 3 base pairs, repair of both mismatches can be depressed (42). Similarly, repair was severely inhibited when nonrepetitive 24-32 nucleotide loop heterologies were placed 33 or 46 base pairs from a base-base mismatch, but repair was efficient when spacing was increased to 325 base pairs (41). MMR involves excision tracts that can extend to hundreds of nucleotides (4), possibly allowing efficient corepair of a nonrepairable mismatch or ID loop when separated from another mismatch by 107 or 1,448 base pairs (43).…”

Nick-Directed Repair of Palindromic Loop Mismatches in Human Cell Extracts

“…The phages used to create the hairpin substrates were constructed by insertion of a 50-base pair palindromic oligonucleotide into the HindIII cleavage site of f1P [44] disrupting the original HindIII site (Figure 1). Two nucleotide randomized positions residing in four overlapping restriction endonuclease recognition sites of the linker allowed for screening mutants by restriction analysis of replication form (RF) DNA.…”

“…Heteroduplexes of base-base mismatch 3¢-AA, and random large loop 5¢-C32, and 5¢-V32 were as described [44].…”