Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro.
BACKGROUND
The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading- and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA.


RESULTS
A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site, but did not affect the progression of the replication fork or leading-strand DNA synthesis. In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis. However, about two-thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced.


CONCLUSIONS
The blocking DNA lesion affects DNA replication differently depending on which strand, leading or lagging, contains the lesion.
Higuchi
Kumiko
K
Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama-cho 8916-5, Ikoma, Nara 630-0101, Japan.
Katayama
Tsutomu
T
Iwai
Shigenori
S
Hidaka
Masumi
M
Horiuchi
Takashi
T
Maki
Hisaji
H
D001483:Base Sequence; D004247:DNA; D004249:DNA Damage; D004258:DNA Polymerase III; D019915:DNA Primase; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics*; D003853:Deoxyribonucleosides / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex; D010957:Plasmids; D018741:Replication Origin; D013698:Templates, Genetic; D014764:Viral Proteins / Q000235:genetics*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003853:Deoxyribonucleosides; C022262:Okazaki fragments; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D014764:Viral Proteins; D004247:DNA; D019915:DNA Primase; D004258:DNA Polymerase III