Single molecule analysis of DNA replication

Herrick, John H.; Bensimon, Aaron

doi:10.1016/s0300-9084(99)00210-2

Cited by 75 publications

(44 citation statements)

References 77 publications

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“…The detection of areas undergoing replication in isolated DNA fibers was originally performed by Bensimon (40,41) and later modified by Jackson and Pombo (42) to generate DNA fibers directly from lysed cells instead of using purified DNA. The DNA fiber extension methodology used in this paper is a modified version of the protocol initially described by Merrick et al (43), which is a modification of Jackson and Pombo's method.…”

Section: Dna Labeling and Fiber Spreadingmentioning

confidence: 99%

Abasic sites preferentially form at regions undergoing DNA replication

et al. 2010

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We investigated whether apurinic/apyrimidinic (AP/abasic) sites were more frequent in regions of DNA replication in cells and whether their number increased during oxidative stress. DNA fiber spreading and fluorescent immunostaining were used to detect areas of DNA replication and sites of AP lesions in extended DNA fibers. The distribution of AP sites was determined in DNA fibers from vertebrate cells maintained under normal culture conditions or stressed with exogenous H(2)O(2). AP lesions per unit length were enumerated in bulk DNA or at replication sites. The background density of AP sites in DNA fibers was 5.4 AP sites/10(6) nt, while newly replicated DNA contained 12.9 AP sites/10(6) nt. In cells exposed to 20 μM H(2)O(2), AP sites in newly replicated DNA increased to 20.8/10(6) nt. Determinations of AP site density in bulk DNA by fiber analysis or standard slot blot assays agreed to within 10%. Our findings show that the fiber assay not only accurately determines the frequency of AP sites but also shows their distribution. They also reveal that there is increased susceptibility to oxidative damage in DNA regions undergoing replication, which may explain the previously observed clustering of AP sites.

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Section: Dna Labeling and Fiber Spreadingmentioning

confidence: 99%

Abasic sites preferentially form at regions undergoing DNA replication

et al. 2010

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“…[8,9] In these studies, a water droplet containing DNA in solution is placed on a surface, such as silanised glass. DNA molecules adhere to the surface by one or both of their ends.…”

Section: Stretching and Twisting Dnamentioning

confidence: 99%

Deforming DNA: From Physics to Biology

2009

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The DNA double helix has become a modern icon which symbolizes our understanding of the molecular basis of life. It is less widely recognized that the double helix proposed by Watson and Crick more than half a century ago is a remarkably adaptable molecule that can undergo major conformational rearrangements without being irreversibly damaged. Indeed, DNA deformation is an intrinsic feature of many of the biological processes in which it is involved. Over the last two decades, single-molecule experiments coupled with molecular modeling have transformed our understanding of DNA flexibility, while the accumulation of high-resolution structures of DNA-protein complexes have demonstrated how organisms can exploit this property as a useful feature for preserving, reading, replicating, and packaging the genetic message. In this Minireview we summarize the information now available on the extreme--and the less extreme--deformations of the double helix.

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“…On the other hand, it is only in the last few years that large amounts of data on the genome-wide coordination have become available. In particular, a technique called molecular combing has been used to examine the replication state of large fractions of the genome by controlled stretching of fluorescently labeled replicated and unreplicated regions onto a substrate [7,8].…”

Section: Introductionmentioning

confidence: 99%

HowXenopus laevisembryos replicate reliably: Investigating the random-completion problem

Yang

Bechhoefer

2008

Phys. Rev. E

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DNA synthesis in Xenopus frog embryos initiates stochastically in time at many sites (origins) along the chromosome. Stochastic initiation implies fluctuations in the time to complete and may lead to cell death if replication takes longer than the cell cycle time (≈ 25 min). Surprisingly, although the typical replication time is about 20 min, in vivo experiments show that replication fails to complete only about 1 in 300 times. How is replication timing accurately controlled despite the stochasticity? Biologists have proposed two solutions to this "random-completion problem." The first solution uses randomly located origins but increases their rate of initiation as S phase proceeds, while the second uses regularly spaced origins. In this paper, we investigate the randomcompletion problem using a type of model first developed to describe the kinetics of first-order phase transitions. Using methods from the field of extreme-value statistics, we derive the distribution of replication-completion times for a finite genome. We then argue that the biologists' first solution to the problem is not only consistent with experiment but also nearly optimizes the use of replicative proteins. We also show that spatial regularity in origin placement does not alter significantly the distribution of replication times and, thus, is not needed for the control of replication timing.

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Single molecule analysis of DNA replication

Cited by 75 publications

References 77 publications

Abasic sites preferentially form at regions undergoing DNA replication

Abasic sites preferentially form at regions undergoing DNA replication

Deforming DNA: From Physics to Biology

HowXenopus laevisembryos replicate reliably: Investigating the random-completion problem

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