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

Yao-Ming Huang,Shee-Uan Chen,Steven D Goodman,Shang-Hsin Wu,Jau-Tsuen Kao,Chun-Nan Lee,Wern-Cherng Cheng,Keh-Sung Tsai,Woei-Horng Fang

doi:10.1074/jbc.m401675200

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.

Highlights

In human cells, large DNA loop heterologies are repaired through a nick-directed pathway independent of mismatch repair
We found a negative interference between 3Ј-nick-directed large loop repair and conventional mismatch repair systems when a mispair is in the vicinity of a loop
We clearly show that human cell extracts can efficiently process looped heteroduplexes containing 12– 429 unpaired nucleotides with a strand break 3Ј to the heterology

Summary

EXPERIMENTAL PROCEDURES

Materials—E. coli strains NM522 and RS5033, bacteriophage f1MR1, and the nucleotide numbering at restriction endonuclease cleavage sites have been described (11, 27). A 24-bp fragment from phage M13mp XbaI and HindIII digestion was inserted into f1MR1 at the HindIII and XbaI cleavage sites (coordinate 5821 and 5832 of f1MR1). The phage in this construction, which retains the HindIII site, is designated f1PM. Substrates containing a nick at the replication origin of the viral strand were prepared by cleaving covalently closed circular heteroduplexes with bacteriophage fd gene II protein (gpII) as described (31). The repair reaction using concentrated nuclear extracts was carried out in 40 ␮l containing 0.02 M Tris-HCl (pH 7.6), 5 mM MgCl2, 0.11 M KCl (derived by titrating extracts with KCl to determine the optimal concentration), 50 ␮g/ml bovine serum albumin, 1 mM ATP, 0.1 mM dATP, 0.1 mM dGTP, 0.1 mM dTTP, 0.1 mM dCTP, and 0.4 ␮g (92 fmol) of heteroduplex DNA. The ethidium complexes of DNA products were quantified using a gel documentation CCD camera (UVP Inc.) (11)

RESULTS

NheI G

TABLE III Efficiency of mismatch repair in HeLa nuclear extracts

Loop o

DISCUSSION