Processivity of T4 endonuclease V is sensitive to sodium chloride concentration

Ganesan, Ann K.; Seawell, Patricia C.; Lewis, Roger J.; Hanawalt, Philip C.

doi:10.1021/bi00367a060

Cited by 76 publications

(40 citation statements)

References 20 publications

(26 reference statements)

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“…A number of proteins involved in DNA repair have been shown to act processively. These include DNA helicases [10], T4 endonuclease V [11,12], Micrococcus luteus UV endonuclease [13], UvrABC nuclease [14], DNA glycosylases [15,16], and AP endonucleases [17].…”

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confidence: 99%

Domains in the XPA protein important in its role as a processivity factor

Bartels

Lambert

2007

Biochemical and Biophysical Research Communications

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XPA is a protein essential for nucleotide excision repair (NER) where it is thought to function in damage recognition/verification. We have proposed an additional role, that of a processivity factor, conferring a processive mechanism of action on XPF and XPG, the endonucleases involved in NER. The present study was undertaken to examine the domain(s) in the XPA gene that are important for the ability of the XPA protein to function as a processivity factor. Using site-directed mutagenesis, mutations were created in several of the exons of XPA and mutant XPA proteins produced. The results showed that the DNA binding domain of XPA is critical for its ability to act as a processivity factor. Mutations in both the zinc finger motif and the large basic cleft in this domain eliminated the ability of XPA to confer a processive mechanism of action on the endonucleases involved in NER.Keywords xeroderma pigmentosum; XPA protein; DNA repair; processive mechanism of action Though the XPA protein is a key component in NER, its precise function in this repair pathway is not clear. There is evidence that it plays a role in the initial steps of NER, where it is involved in damage recognition/verification [1][2][3]. We have proposed an additional important function for XPA, that of a processivity factor needed to confer a processive mechanism of action on the endonucleases, XPF and XPG, involved in NER [4,5]. Proteins can locate target sites on DNA by two distinctive mechanisms: (1) a processive mechanism in which a protein first binds to a random site on DNA and then translocates to a specific site by a facilitated-diffusion process in which it slides or hops along the DNA; or (2) a distributive mechanism, in which a protein has no affinity for non-target DNA and locates target sites by a random, threedimensional diffusion process [6][7][8][9]. The mechanism utilized by DNA-targeting proteins is extremely important in determining the ability of the protein to properly interact with its target sites on DNA and, for many of these proteins, a processive mechanism of action is essential [6,8] We have previously shown that during repair of UVC light induced cyclobutane pyrimidine dimers in normal human cells, the endonucleases, XPG and XPF, incise DNA at sites of damage using a processive mechanism of action [4,5]. In contrast, these endonucleases in cells from

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confidence: 99%

Domains in the XPA protein important in its role as a processivity factor

Bartels

Lambert

2007

Biochemical and Biophysical Research Communications

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“…Apparently, the W128S mutation is more pronounced at 10 mM than at 100 mM salt concentration. One interpretation of these results is that tryptophan-128 is involved in DNA processivity which is somehow functionally coupled to the nicking activity ofthe AP lyase activity since both these processes are more evident at lower salt concentrations (36,37). An oligonucleotide with a single abasic site may not clearly distinguish the defect of W128S if this mutation involves DNA processivity.…”

Section: Resultsmentioning

confidence: 94%

“…One possible explanation for these discrepancies may be the conditions for assaying endonuclease V activity. Lantham et al (24) used salt concentrations of 100mM which favors a distributive action of the enzyme (36,37) and which suppresses the AP lyase activity (5). At low salt concentrations (10 mM), which was used in our study and in several previous studies (3,5), the AP lyase is stimulated.…”

Section: Resultsmentioning

confidence: 99%

“…However, because a much lower concentration of this substrate was used than in the poly(dA)-poly(dT) assay, we cannot rule out that this difference is due to a Km effect. It is possible that the W128S mutation affects both the N-glycosylase and AP lyase by a mechanism which does not involve catalytic function but some other common denominator for efficient damage incision, such as CBPD binding (22) or DNA processivity (36,37). Apparently, the W128S mutation is more pronounced at 10 mM than at 100 mM salt concentration.…”

Section: Resultsmentioning

confidence: 99%

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Replacing tryptophan-128 of T4 endonuclease V with a serine residue results in decreased enzymatic activityin vitroandin vivo

Valerie

1995

Nucl Acids Res

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“…We were able to effectively map the DNA repair domain in the DHFR region in CHO cells by analysis of the efficiency of pyrimidine dimer removal from a series of overlapping restriction fragments, 7 to 30 kb in length, which spanned the entire gene and its surrounding sequences (19). We found that within 8 hr after a UV dose of 20 J/m2, the cells had removed more than 40% (20,21).…”

Section: Introductionmentioning

confidence: 82%