Abstract
DNA loop heterologies are products of normal DNA metabolism and can lead to severe genomic instability if unrepaired. To understand how human cells process DNA loop structures, a set of circular heteroduplexes containing a 30-nucleotide loop were constructed and tested for repair in vitro by human cell nuclear extracts. We demonstrate here that, in addition to the previously identified 5' nick-directed loop repair pathway (Littman, S. J., Fang, W. H., and Modrich, P. (1999) J. Biol. Chem. 274, 7474-7481), human cells can process large DNA loop heterologies in a loop-directed manner. The loop-directed repair specifically removes the loop structure and occurs only in the looped strand, and appears to require limited DNA synthesis. Like the nick-directed loop repair, the loop-directed repair is independent of many known DNA repair pathways, including DNA mismatch repair and nucleotide excision repair. In addition, our data also suggest that an aphidicolin-sensitive DNA polymerase is involved in the excision step of the nick-directed loop repair pathway.
Highlights
DNA loop heterologies are unpaired, single-stranded DNA structures that can be generated during DNA metabolism
The processing of small DNA loops with a strand break 5Ј to the heterology occurs by a manner similar to that seen for mismatch repair (MMR)-dependent processing, the processing of looped heteroduplexes containing a 3Ј strand break seems to involve endonuclease(s) that remove the loop directly, without excision occurring from the nick [20]
The results presented here demonstrate that there are at least two distinct repair pathways for large DNA loops in human cells: one that is directed by a strand break and the other that is directed by the loop itself
Summary
Cell Culture and Nuclear Extract Preparation—HeLa cells were grown in RPMI 1640 with 5% fetal bovine serum and 4 mM glutamine. 5Ј-30V describes a substrate with a 5Ј nick and 30-nt loop in the viral (V) strand (see Fig. 1). Reaction Intermediate Analysis—Reaction intermediates were trapped during in vitro repair assays by the omission of exogenous dNTPs, the addition of aphidicolin, or the addition of ddNTPs. Purified reaction products were digested with SspI, separated on 6% denaturing polyacrylamide gels, and electrotransferred onto a nylon membrane as described [20]. To probe for single stranded DNA regions between the nick and the loop, repair intermediates trapped by limited DNA synthesis were alternately digested with 2 units each of BseRI and BanII (scoring enzyme). Band intensities were analyzed by digital photography using Kodak Image 2.0.2 software
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