Inhibition of the 5′ to 3′ exonuclease activity of hEXO1 by 8‐oxoguanine

McDowell, Heather D.; Carney, James; Wilson, Teresa

doi:10.1002/em.20398

Cited by 5 publications

(2 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hexahistidine-tagged recombinant human EXO1, overexpressed in insect cells and purified as described elsewhere [30], was kindly provided by Dr. T. Wilson (University of Maryland School of Medicine). Hexahistidine-tagged WRN was overexpressed in insect cells and purified as described elsewhere [31].…”

Section: Methodsmentioning

confidence: 99%

Delineation of WRN helicase function with EXO1 in the replicational stress response

et al. 2010

View full text Add to dashboard Cite

The WRN gene defective in the premature aging disorder Werner syndrome encodes a helicase/ exonuclease. We examined the ability of WRN to rescue DNA damage sensitivity of a yeast mutant defective in the Rad50 subunit of Mre11-Rad50-Xrs2 nuclease complex implicated in homologous recombination repair. Genetic studies revealed WRN operates in a yEXO1-dependent pathway to rescue rad50 sensitivity to methylmethane sulfonate (MMS) and prevent mitotic catastrophe. WRN helicase, but not exonuclease, is required for MMS resistance. WRN missense mutations in helicase or RecQ C-terminal domains interfered with the ability of WRN to rescue rad50 MMS sensitivity. WRN does not rescue rad50 ionizing radiation (IR) sensitivity, suggesting that WRN, in collaboration with yEXO1, is tailored to relieve replicational stress imposed by alkylated base damage. WRN and yEXO1 are associated with each other in vivo. Purified WRN stimulates hEXO1 nuclease activity on DNA substrates associated with a stalled or regressed replication fork. We propose WRN helicase operates in an EXO1-dependent pathway to help cells survive replicational stress. In contrast to WRN, BLM helicase defective in Bloom's syndrome failed to rescue rad50 MMS sensitivity, but partially restored IR resistance, suggesting a delineation of function by the human RecQ helicases.

show abstract

Section: Methodsmentioning

confidence: 99%

Delineation of WRN helicase function with EXO1 in the replicational stress response

et al. 2010

View full text Add to dashboard Cite

show abstract

“…The annealed duplex with a 6-nucleotide overhang at the 5’ end of the G -strand were labeled at the 3’ end with [α- 32 P]dATP as described [62]. The assays were performed with 5 or 10 nM purified hMutSα and various amounts of purified h9-1-1 complex, Sp9-1-1 complex, hHus1, hRad1, or hRad9 as denoted in the figure legend in 20 µl volume containing 5 fmol DNA substrate, 100 mM NaCl, 25 mM HEPES-NaOH (pH8.0), 1 mM MgCl 2 , 0.1 mM ADP, 1 mM dethiothreitol, 0.1 mM EDTA, 10% glycerol, 0.075 mg/ml bovin serum albumin (BSA) and 150 ng of 200 base pair homoduplex competitor [63]. The reactions were incubated at 37°C for 20 min and separated on a 5% polyacrylamide gel containing 2.5% glycerol.…”

Section: Methodsmentioning

confidence: 99%

Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1

et al. 2010

View full text Add to dashboard Cite

In eukaryotic cells, the cell cycle checkpoint proteins Rad9, Rad1, and Hus1 form the 9-1-1 complex which is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp. hMSH2/hMSH6 (hMutSα) and hMSH2/hMSH3 (hMutSβ) are the mismatch recognition factors of the mismatch repair pathway. hMutSα has been shown to physically and functionally interact with PCNA. Moreover, DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment induces the G2/M cell cycle arrest that is dependent on the presence of hMutSα and hMutLα. In this study, we show that each subunit of the human 9-1-1 complex physically interacts with hMSH2, hMSH3, and hMSH6. The 9-1-1 complex from both humans and Schizosaccharomyces pombe can stimulate hMutSα binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutSα. Human Rad9 and hMSH6 colocalize to nuclear foci of HeLa cells after exposure to MNNG. However, Rad9 does not form foci in MSH6 defective cells following MNNG treatment. In Rad9 knockdown untreated cells, the majority of the MSH6 is in cytoplasm. Following MNNG treatment, Rad9 knockdown cells has abnormal nuclear morphology and MSH6 is distributed around nuclear envelop. Our findings suggest that the 9-1-1 complex is a component of the mismatch repair involved in MNNG-induced damage response.

show abstract

DNA repair and the origins of urinary oxidized 2'-deoxyribonucleosides

2010

View full text Add to dashboard Cite

Monitoring oxidative stress in vivo is made easier by the ability to use samples obtained non-invasively, such as urine. The analysis of DNA oxidation, by measurement of oxidized 2'-deoxyribonucleosides in urine, particularly 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), has been reported extensively in the literature in many situations relating to various pathologies, populations and environmental exposures. Understanding the origins of urinary 8-oxodG, other than it simply being a marker of DNA oxidation or its synthetic precursors, is important to being able to effectively interpret differences in baseline urinary 8-oxodG levels between subject groups and changes in excretion. Diet and cell turnover play negligible roles in contributing to urinary 8-oxodG levels, leaving DNA repair as a primary source of this lesion. However, which repair processes contribute, and to what extent, to urinary 8-oxodG is still open to question. The most rational source would be the activity of selected members of the Nudix hydrolase family of enzymes, sanitizing the deoxyribonucleotide pool via the degradation of 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-triphosphate and 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-diphosphate, yielding mononucleotide products that can then be dephosphorylated to 8-oxodG and excreted. However, nucleotide excision repair (NER), transcription-coupled repair, nucleotide incision repair (NIR), mismatch repair and various exonuclease activities, such as proofreading function associated with DNA polymerases, can all feasibly generate initial products that could yield 8-oxodG after further metabolism. A recent study implying that a significant proportion of genomic 8-oxodG exists in the context of tandem lesions, refractory to repair by glycosylases, suggests the roles of NER and/or NIR remain to be further examined and defined as a source of 8-oxodG. 8-OxodG has been the primary focus of investigation, but other oxidized 2'-deoxyribonucleosides have been detected in urine, 2'-deoxythymidine glycol and 5-hydroxymethyl-2'-deoxyuridine; the origins of these compounds in urine, however, are presently even more speculative than for 8-oxodG.

show abstract

Inhibition of the 5′ to 3′ exonuclease activity of hEXO1 by 8‐oxoguanine

Cited by 5 publications

References 55 publications

Delineation of WRN helicase function with EXO1 in the replicational stress response

Delineation of WRN helicase function with EXO1 in the replicational stress response

Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1

DNA repair and the origins of urinary oxidized 2'-deoxyribonucleosides

Contact Info

Product

Resources

About