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Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro
Kumiko Higuchi1, Tsutomu Katayama, Shigenori Iwai
1Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama-cho 8916-5, Ikoma, Nara 630-0101, Japan.
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.
Insights
DNA lesions impede DNA replication differently based on strand location. Lesions on the lagging strand block Okazaki fragment synthesis, while leading strand lesions stall replication forks and inhibit synthesis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA replication fork progression is crucial for genome stability.
- DNA lesions can inhibit replication, leading to cell death or instability.
- The impact of DNA lesions on concurrent leading- and lagging-strand synthesis is not well understood.
Purpose of the Study:
- To investigate how DNA lesions affect replication fork dynamics.
- To examine the differential impact of lesions on leading vs. lagging strand synthesis.
Main Methods:
- Utilized a semi-bidirectional DNA replication system with purified Escherichia coli enzymes.
- Employed an oriC plasmid with a replication-terminating protein.
- Introduced a single abasic DNA lesion on the template DNA.
Main Results:
- A lesion on the lagging strand blocked Okazaki fragment synthesis but not fork progression or leading-strand synthesis.
- A lesion on the leading strand stalled the replication fork and inhibited leading-strand synthesis.
- Leading strand lesions allowed lagging-strand synthesis beyond the lesion in two-thirds of cases, albeit at reduced fork velocity.
Conclusions:
- DNA lesions differentially impact DNA replication based on their location on the leading or lagging strand.
- Strand-specific lesion effects highlight the complex coordination of replication machinery.