A specific 3' exonuclease activity of UvrABC

Gordienko, Irina; Rupp, W. Dean

doi:10.1093/emboj/17.2.626

Cited by 21 publications

(18 citation statements)

References 53 publications

(74 reference statements)

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“…This three-base strand separation was positioned from the modified nucleotide through the second base 3′ to the adduct. The result for AAF is generally consistent with that of a previous study (29). Taken together, our results demonstrated the dependence of strand opening on the type of DNA adduct during DNA damage recognition.…”

Section: Discusssionsupporting

confidence: 92%

“…It has been previously reported that the second 5′-incision is coupled with the first 5′-incision (12), so that the second incisions can occur only after the primary incision. It is believed that this second 5′-incision might be the result of a damage-independent endonuclease activity of UvrABC or UvrBC that makes incisions at the seventh and eighth phosphodiester bonds 5′ to a nicked site of nondamaged DNA (just like the product of the first 5′-incision) (39,40). Since the top strand of flap substrates had been 5′-radiolabeled with 32 P, the second incision made the prior incision invisible in the gel.…”

Section: Resultsmentioning

confidence: 99%

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Effects of DNA Adduct Structure and Sequence Context on Strand Opening of Repair Intermediates and Incision by UvrABC Nuclease

et al. 2003

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DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N 2 -guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.Nucleotide excision repair (NER), 1 as one of the primary DNA repair pathways in cells, is capable of removing an extensive variety of bulky lesions, induced by chemicals and radiation, with varying efficiencies (1-3). Its ability to recognize and excise such a broad repertoire of substrates has been the subject of intensive research, and it is generally believed that the success of NER depends on efficient damage recognition. The UvrABC nuclease system, which initiates the NER in Escherichia coli, represents a paradigm for understanding the general mechanism of DNA damage recognition and incision (1). This model system has been widely

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Section: Discusssionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Effects of DNA Adduct Structure and Sequence Context on Strand Opening of Repair Intermediates and Incision by UvrABC Nuclease

et al. 2003

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“…Additional cutting was recently shown to be related to a damage-independent incision activity of the UvrABC proteins on a substrate containing a single strand-double strand junction (15). Another damageindependent incision activity of the Uvr proteins was reported by Zou et al (16).…”

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confidence: 76%

Characterization of the Escherichia coliDamage-independent UvrBC Endonuclease Activity

Moolenaar

Bazuine

Knippenberg

et al. 1998

Journal of Biological Chemistry

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Incision of damaged DNA templates by UvrBC in Escherichia coli depends on UvrA, which loads UvrB on the site of the damage. A 50-base pair 3 prenicked DNA substrate containing a cholesterol lesion is incised by UvrABC at two positions 5 to the lesion, the first incision at the eighth and the second at the 15th phosphodiester bond. Analysis of a 5 prenicked cholesterol substrate revealed that the second 5 incision is efficiently produced by UvrBC independent of UvrA. This UvrBC incision was also found on the same substrate without a lesion and, with an even higher efficiency, on a DNA substrate containing a 5 single strand overhang. Incision occurred in the presence of ATP or ADP but not in the absence of cofactor. We could show an interaction between UvrB and UvrC in solution and subsequent binding of this complex to the substrate with a 5 single strand overhang. Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activity requires the protein-protein interaction domains, which are exclusively needed for the 3 incision on damaged substrates. However, the UvrBC incision uses the catalytic site in UvrC which makes the 5 incision on damaged DNA substrates.In Escherichia coli nucleotide excision repair is initiated by the UvrA, UvrB, and UvrC proteins. Sequential action of these three proteins leads to two incisions in the damage-containing DNA strand, one on either side of the lesion (1-3). In solution a UvrA 2 B complex is formed, in which UvrA is the damagerecognizing subunit (4). After initial binding of the trimeric protein complex to the site of a DNA lesion, UvrB is loaded onto the DNA, forming a stable UvrB⅐DNA preincision complex (5). The actual incisions take place in a complex consisting of DNA, UvrB, and UvrC. The first incision is made at the third, fourth, or fifth phosphodiester bond 3Ј of the lesion, followed by an incision at the eighth phosphodiester bond 5Ј of the lesion (6). Several observations indicate that the conformation of the UvrBC⅐DNA complex differs for the 3Ј and 5Ј incision reactions. First of all, the 5Ј incision can only take place when a nick is present at the 3Ј incision position, introduced either enzymatically or artificially (7,8). This strongly suggests that the DNA adopts a different conformation in the two complexes. A second observation is that the 3Ј incision requires the interaction between the C-terminal domain of UvrB and a homologous internal domain of UvrC, whereas this interaction is dispensable for the 5Ј incision (8, 9). Conversely, the 5Ј incision requires the presence of a DNA binding domain located in the C-terminal part of UvrC, whereas this domain can be omitted for the 3Ј incision (10). In the initial UvrBC⅐DNA complex the catalytic site for 3Ј incision is most likely positioned at the scissile phosphodiester bond, incision of which triggers a transition in the complex positioning the catalytic site for the 5Ј incision.

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“…It is not clear from our experiments whether the same incision activity on nondamaged DNA has a function in other processes in the cell. DNA incision by a complex of UvrB and UvrC in the absence of DNA damage has also been shown in vitro (9,28,48). This incision, however, which takes place seven nucleotides from a single strand-double strand junction, is induced by the catalytic site that on damaged DNA makes the 5Ј nick and is independent of UvrA (28).…”

Section: Vol 182 2000 Role Of Ner Proteins In Dna Replication 5711mentioning

confidence: 88%

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

2000

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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...

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A specific 3' exonuclease activity of UvrABC

Cited by 21 publications

References 53 publications

Effects of DNA Adduct Structure and Sequence Context on Strand Opening of Repair Intermediates and Incision by UvrABC Nuclease

Effects of DNA Adduct Structure and Sequence Context on Strand Opening of Repair Intermediates and Incision by UvrABC Nuclease

Characterization of the Escherichia coliDamage-independent UvrBC Endonuclease Activity

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

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