Properties of Bacteriophage T4 Proteins Deficient in Replication Repair

Abstract

An epistasis group of mutations engendering increased sensitivity to diverse DNA-damaging agents was described previously in bacteriophage T4. These mutations are alleles of genes 32 and 41, which, respectively, encode a single-stranded DNA-binding protein (gp32) and the replicative DNA helicase (gp41). The mechanism by which the lethality of DNA damage is mitigated is unknown but seems not to involve the direct reversal of damage, excision repair, conventional recombination repair, or translesion synthesis. Here we explore the hypothesis that the mechanism involves a switch in DNA primer extension from the cognate template to an alternative template, the just-synthesized daughter strand of the other parental strand. The activities of the mutant proteins are reduced about 2-fold (for gp32) or 4-fold (for gp41) in replication complexes catalyzing coordinated synthesis of leading and lagging strands, in binding single-stranded DNA, promoting DNA annealing, and promoting branch migration. In striking contrast, the mutant proteins are strongly impaired in promoting template switching, thus supporting the hypothesis of survival by template switching.Four general mechanisms that either repair or circumvent potentially lethal damage to DNA have been extensively investigated. These are (i) direct reversal of the damage, as in photoreactivation or dealkylation, (ii) damage excision followed by resynthesis templated by the complementary strand, as in base excision repair and nucleotide excision repair, (iii) recombinational repair, in which the damaged DNA obtains the requisite sequence information from another chromosome or from the sister chromosome in a way that depends on a recombinase and a Holliday-junction resolvase, and (iv) translesion synthesis, which is frequently mutagenic. Strong hints of a fifth mechanism have accumulated over time, but a combined genetic and enzymological demonstration of such a mechanism has not been available. These hints accumulated in two parallel lines of investigation, one using eukaryotic cells and the other using bacteriophage T4. Both lines point toward a repair mechanism based on a template-switching process operating directly at the replication fork.In a classic model of the fifth mechanism (1), DNA replication on one parental template strand is blocked by a lesion while replication on the other parental strand continues briefly, whereupon strand displacement and branch migration re-associate the two daughter strands into a short duplex (Fig. 1). The blocked daughter strand then continues replication on the alternative template, and then the structure reforms a conventional replication fork with the lesion having been bypassed. This model was supported by experiments in which bromodeoxyuridine was used as a "heavy" label and tritiated thymidine as a "light" label during the replication of DNA in UV-irradiated mammalian cells: a small amount of tritiated DNA of "heavy-heavy" density was observed in DNA from irradiated cells, and this was chased into "heavy-light" DNA. In addition...

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

“…The lesion consists of an abasic site that is followed immediately by a template base (G) that cannot easily serve as a template because the reaction mixture is devoid of dCTP (which is needed nowhere else in the reaction). This double lesion effectively abolishes translesion synthesis (7). The other key feature of the DNA substrate is that the second switch is likely to start when the extension of strand 3 reaches or penetrates the template pentaribonucleotide 5Ј-rArCrCrUrU-3Ј, which is characteristic of the primers that initiate lagging strands in T4 replication forks (25,26).…”

“…2); Dda can be replaced by the gp41 replicative helicase plus its gp59 loader, although inefficiently. A similar but single strandswitching process can be driven not by UvsX plus Dda but by the gp41 helicase plus the gp32 single-stranded DNA-binding protein (7). However, this process has been examined so far only using a replication fork designed in a way that prevented the second switch.…”

“…Proteins-Overproduction and purification of phage T4 gp32, gp43, gp43D219A, gp44/62, gp59, UvsX, and UvsY were as described previously (7,24). gp41 and gp45 were purified as described previously (24) with modifications to be described elsewhere.…”

“…We included gp59 in the analysis because it loads the gp41 helicase onto DNA (27) and is required for single template switching driven by gp41 (7). When gp41 and gp59 were added to a reaction also containing UvsX and the polymerase holoenzyme proteins, the 120-nt doubleswitching product accumulated (Fig.…”

“…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).…”