PMID- 10373362
OWN - NLM
STAT- MEDLINE
DCOM- 19990728
LR  - 20071114
IS  - 0022-2836 (Print)
IS  - 0022-2836 (Linking)
VI  - 289
IP  - 5
DP  - 1999 Jun 25
TI  - Analysis of DNA replication forks encountering a pyrimidine dimer in the template
      to the leading strand.
PG  - 1207-18
AB  - Electron microscopy (EM) was used to visualize intermediates of in vitro
      replication of closed circular DNA plasmids. Cell-free extracts were prepared
      from human cells that are proficient (IDH4, HeLa) or deficient (CTag) in bypass
      replication of pyrimidine dimers. The DNA substrate was either undamaged or
      contained a single cis, syn thymine dimer. This lesion was inserted 385 bp
      downstream from the center of the SV40 origin of replication and sited
      specifically in the template to the leading strand of the newly synthesized DNA. 
      Products from 30 minute reactions were crosslinked with psoralen and UV,
      linearized with restriction enzymes and spread for EM visualization. Extended
      single-stranded DNA regions were detected in damaged molecules replicated by
      either bypass-proficient or deficient extracts. These regions could be coated
      with Escherichia coli single-stranded DNA binding protein. The length of duplex
      DNA from a unique restriction site to the single-stranded DNA region was that
      predicted from blockage of leading strand synthesis by the site-specific dimer.
      These results were confirmed by S1nuclease treatment of replication products
      linearized with single cutting restriction enzymes, followed by detection of the 
      diagnostic fragments by gel electrophoresis. The absence of an extended
      single-stranded DNA region in replication forks that were clearly beyond the
      dimer was taken as evidence of bypass replication. These criteria were fulfilled 
      in 17 % of the molecules replicated by the IDH4 extract.
CI  - Copyright 1999 Academic Press.
FAU - Cordeiro-Stone, M
AU  - Cordeiro-Stone M
AD  - Department of Pathology, University of North Carolina, Chapel Hill, USA.
      uncmcs@med.unc.edu
FAU - Makhov, A M
AU  - Makhov AM
FAU - Zaritskaya, L S
AU  - Zaritskaya LS
FAU - Griffith, J D
AU  - Griffith JD
LA  - eng
GR  - CA55065/CA/NCI NIH HHS/United States
GR  - GM31819/GM/NIGMS NIH HHS/United States
GR  - S-P30-CA16086/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - J Mol Biol
JT  - Journal of molecular biology
JID - 2985088R
RN  - 0 (DNA, Circular)
RN  - 0 (DNA, Single-Stranded)
RN  - 0 (DNA, Viral)
RN  - 0 (Pyrimidine Dimers)
SB  - IM
MH  - Cell Line, Transformed
MH  - *DNA Replication
MH  - DNA, Circular/physiology/ultrastructure
MH  - DNA, Single-Stranded/ultrastructure
MH  - DNA, Viral/*physiology/ultrastructure
MH  - HeLa Cells
MH  - Humans
MH  - *Pyrimidine Dimers
MH  - *Replication Origin
MH  - Simian virus 40/*genetics/physiology
MH  - Templates, Genetic
MH  - Virus Replication
EDAT- 1999/06/22 00:00
MHDA- 1999/06/22 00:01
CRDT- 1999/06/22 00:00
PHST- 1999/06/22 00:00 [pubmed]
PHST- 1999/06/22 00:01 [medline]
PHST- 1999/06/22 00:00 [entrez]
AID - 10.1006/jmbi.1999.2847 [doi]
AID - S0022-2836(99)92847-4 [pii]
PST - ppublish
SO  - J Mol Biol. 1999 Jun 25;289(5):1207-18. doi: 10.1006/jmbi.1999.2847.