Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand

Electron microscopy (EM) was used to visualize intermediates of in vitro re plication of closed circular DNA plasmids. Cell-free extracts were prepared from human cells that are proficient (IDH4, HeLa) or deficient (CTag) in b ypass replication of pyrimidine dimers. The DNA substrate was either undama ged or contained a single cis,syn thymine dimer. This lesion was inserted 3 85 bp downstream from the center of the SV40 origin of replication and site d specifically in the template to the leading strand of the newly synthesiz ed DNA. Products from 30 minute reactions were crosslinked with psoralen an d UV, linearized with restriction enzymes and spread for EM visualization. Extended single-stranded DNA regions were detected in damaged molecules rep licated by either bypass-proficient or deficient extracts. These regions co uld be coated with Escherichia coli single-stranded DNA binding protein. Th e length of duplex DNA from a unique restriction site to the single-strande d DNA region was that predicted from blockage of leading strand synthesis b y the site-specific dimer. These results were confirmed by S, nuclease trea tment of replication products linearized with single cutting restriction en zymes, followed by detection of the diagnostic fragments by gel electrophor esis. The absence of an extended single-stranded DNA region in replication forks that were clearly beyond the dimer was taken as evidence of bypass re plication. These criteria were fulfilled in 17% of the molecules replicated by the IDH4 extract. (C) 1999 Academic Press.