Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

Abstract

DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, ⌬polA cells grow even better when the uvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3 incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the ⌬polA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.In Escherichia coli, nucleotide excision repair (NER) is initiated by the action of the UvrA, UvrB, and UvrC proteins. The UvrA protein loads UvrB onto a damaged site, after which UvrC binds to UvrB, resulting in the UvrBC-DNA incision complex. In this complex, first an incision is made at the fourth or fifth phosphodiester bond on the 3Ј side of the damage, followed by incision at the eighth phosphodiester bond on the 5Ј side of the damage. Both incisions are catalyzed by the UvrC protein, which contains two distinct active sites, one for each incision (20,45). UvrD (helicase II) subsequently removes the damaged strand, and DNA polymerase I (PolI) fills in the resulting gap. Finally, the remaining nick is closed by DNA ligase (for reviews, see references 8 and 36).Besides its function in NER, it is generally believed that the major role of PolI in the cell is the processing of the lagging strand during DNA replication (16). In polA mutant strains the joining of Okazaki fragments is severely retarded (31,32,42). The protein possesses three enzymatic activities, a 5Ј-3Ј exonuclease activity located in the N-terminal part of the protein (the small domain) and a DNA polymerase activity which, together with a 3Ј-5Ј exonuclease activity, is located in the C-terminal part of the protein (the Klenow domain) (5, 15). The combination of the 5Ј-3Ј exonuclease and the polymerase activities results in the so-called nick translation ac...

Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

“…Upon encountering an altered DNA structure UvrA delivers UvrB to the DNA by providing two important functions: 1) UvrA bends the DNA at the site of the damage and helps to open the two strands, and 2) UvrA relieves UvrB's domain 4 autoinhibitory domain thereby activating UvrB's cryptic ATPase allowing transfer of the damaged DNA to UvrB [5][6][7]. UvrB then searches the DNA for the exact site of the damaged nucleotide using aromatic side-chains along a β-hairpin motif that is inserted between the two strands of DNA [8][9][10][11][12]. During this verification step, UvrB takes possession of the DNA causing UvrA to dissociate.…”

Stimulation of UvrD Helicase by UvrAB

“…This alternative pathway(s) of Okazaki fragment processing is poorly defined. However, viability of ⌬polA strains is dependent on the presence of UvrA, -B, and -D (47)(48)(49)(50)(51), suggesting that these three nucleotide excision repair proteins play key roles in this alternative pathway. Moreover, deletion of uvrC enhances the viability of ⌬polA cells implying that UvrC activity inhibits this alternative pathway (51).…”

“…However, viability of ⌬polA strains is dependent on the presence of UvrA, -B, and -D (47)(48)(49)(50)(51), suggesting that these three nucleotide excision repair proteins play key roles in this alternative pathway. Moreover, deletion of uvrC enhances the viability of ⌬polA cells implying that UvrC activity inhibits this alternative pathway (51). These in vivo data were explained by a model in which UvrAB binds to nicks in newly synthesized lagging strand DNA even in the absence of DNA damage (51).…”

“…Moreover, deletion of uvrC enhances the viability of ⌬polA cells implying that UvrC activity inhibits this alternative pathway (51). These in vivo data were explained by a model in which UvrAB binds to nicks in newly synthesized lagging strand DNA even in the absence of DNA damage (51). Promotion of Okazaki fragment processing was proposed to involve conferral of a specific direction on UvrD-catalyzed unwinding by binding of UvrAB, promoting removal of the RNA primers by a presumed nuclease, and subsequent sealing of the gap by a DNA polymerase and ligase.…”

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

“…Although these cells are extremely defective in the joining of Okazaki fragments, an alternative UvrD-dependent replication pathway is hypothesized to remove RNA primers and fill in these gaps. This alternative pathway requires not only UvrD but also UvrA and UvrB (not UvrC) (6,11) such that double mutants of polymerase I and UvrA, -B, or -D are lethal. It is hypothesized that the UvrABD complex can replace the stranddisplacement function of polymerase I, whereas alternative exonucleases and polymerases complete lagging strand DNA synthesis (6).…”