UvsX Recombinase and Dda Helicase Rescue Stalled Bacteriophage T4 DNA Replication Forks in Vitro

Abstract

The rescue of stalled replication forks via a series of steps that include fork regression, template switching, and fork restoration often has been proposed as a major mechanism for accurately bypassing non-coding DNA lesions. Bacteriophage T4 encodes almost all of the proteins required for its own DNA replication, recombination, and repair. Both recombination and recombination repair in T4 rely on UvsX, a RecA-like recombinase. We show here that UvsX plus the T4-encoded helicase Dda suffice to rescue stalled T4 replication forks in vitro. This rescue is based on two sequential template-switching reactions that allow DNA replication to bypass a non-coding DNA lesion in a non-mutagenic manner.DNA template-strand lesions that prevent the extension of primer strands are usually lethal. Four general mechanisms operate to overcome such lethality: direct repair that regenerates a native structure (e.g. photoreaction and dealkylation), excision repair that replaces damaged bases or deoxyribose residues, translesion synthesis that is usually mutation-prone, and recombination repair that accurately bypasses DNA damage.The essence of recombination repair is that it derives accurate genetic information from the nascent sister chromatid (or from a homologous chromosome when repairing a double strand break); excision repair, on the other hand, derives the requisite genetic information from the complementary strand of the same double helix. Recombination repair was first characterized in Escherichia coli (1-4). The classical E. coli breakreunion model holds that blocked primer extension is followed by downstream reinitiation, leaving a gap that is then filled at least in part by the physical donation of a strand of appropriate polarity cut from the sister chromatid. The donating strand gap is filled by DNA synthesis, whereas the blocking lesion remains as a problem for the future.Recent progress in understanding recombination repair, based mainly on work with E. coli, has highlighted its importance for rescuing replication forks stalled at sites of DNA damage or spontaneous fork collapse (8 -11). One model of recombination repair (Fig. 1) invokes a topological rearrangement in which the fork regresses into a configuration in which the parental strands reanneal and the two daughter strands anneal. This rearrangement generates a structure that constitutes a Holliday junction and that to some resembles a chicken foot (12). In the case of a primer strand whose extension is blocked by a lesion in the template strand, fork regression provides a template that allows limited primer extension. Subsequent reformation of a conventional replication fork then provides a primer strand that has accurately bypassed the template lesion. This pathway was named replication repair and was initially suggested to occur during mammalian DNA replication (5). It was later validated by genetical and enzymological analyses in phage T4 (6, 7).In addition to a DNA polymerase, the canonical model of replication repair (Fig. 1) immediately suggests roles f...

In Vivo Evidence for a recA -Independent Recombination Process in Escherichia coli That Permits Completion of Replication of DNA Containing UV Damage in Both Strands

“…More specifically, we wished to investigate whether there was evidence for E. coli possessing a transient template-switching mechanism of the kind first proposed by Higgins, Kato and Strauss in 1976 (6) for mammalian cells and later proposed for E. coli (4,5,16,19,24). In addition, a mechanism of this kind has been reconstructed in vitro, using purified phage T4 proteins (8,9). However, this model has been difficult to explore experimentally in vivo because the recombination event depends on informa-tional, rather than physical, exchange between DNA strands and because it occurs between identical sister strands.…”

“…Because such transfer occurs between genetically identical sister duplexes, exchange cannot be detected by means of genetically marked strains. As a consequence, there is little in vivo evidence to support the occurrence of such a mechanism or to indicate its genetic requirements, such as dependence on RecA, though in vitro results with E. coli and phage T4 proteins strongly support such a model (5,7,8,9,16,17,(19)(20)(21). We have investigated the possible occurrence of such an alternative recombination mechanism for the completion of replication in the face of unrepaired DNA damage by transforming various E. coli strains with plasmids in which each strand carried, at specific staggered positions, a single thyminethymine pyrimidine (6-4) pyrimidinone [T-T (6-4)] lesion.…”

Templated Mutagenesis in Bacteriophage T4 Involving Imperfect Direct or Indirect Sequence Repeats

“…Subsequent enzymological analyses using an eight-protein T4 DNA replication system in vitro showed that strand switching could indeed be promoted by DNA damage in the template strand and that the resulting replication repair was severely compromised when the mutant gp32 and gp41 proteins replaced their wild-type counterparts (Kadyrov and Drake 2003). Then, somewhat surprisingly, an alternative T4 system of replication repair was discovered in vitro in which gp32 and gp41 were replaced by the classical T4 recombinase UvsX and a different T4 DNA helicase, Dda (Kadyrov and Drake 2004). To date, these are the only genetically and enzymologically well-defined replication-repair systems.…”

Hjm/Hel308A DNA Helicase fromSulfolobus tokodaiiPromotes Replication Fork Regression and Interacts with Hjc Endonuclease In Vitro

“…The translesion synthesis pathway is error prone due to the participation of low-fidelity DNA polymerases (21). The cleavage pathway is thought to be the dominant mode in bacteria, while replication repair (through template switching and translesion synthesis) is the dominant mode in T4 and eukaryotes (14).…”