Two components of DNA replication-dependent LexA cleavage

Abstract

Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, single-stranded DNA. RecA binds to single-stranded DNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in vitro in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a single-stranded DNA–RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage similar to that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate single-stranded DNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand single-stranded DNA gaps generated by lesion skipping.

An autonomous mathematical model for the mammalian cell cycle

“…A damping factor depending on the number of actively replicating origins A is included. Active replication forks have single-stranded DNA [131] which binds Replication Protein A, activating the ATR-CHK1 pathway and inhibiting DNA replication. In the absence of any damping, replication of the human genome would take about an hour under optimal nutrient conditions, based on a maximal fork velocity 2 kb/min [136] and an average inter-origin spacing (replicon size) of 125 kb [141], whereas S phase in a normal human cell cycle takes 8-10 hours [142].…”

“…Term 3: Cdc25A is degraded by SCF Trcp when the ATR-CHK1 pathway is activated by Replication Protein A binding singlestranded DNA [42,119,[127][128][129][130]. In a normal unperturbed cell cycle, single-stranded DNA is present when forks are actively replicating [131,132], i.e. when A is nonzero.…”