DNA Adduct Bypass Polymerization by Sulfolobus solfataricus DNA Polymerase Dpo4

Hong, Zhou; Goodenough, Angela K.; Choi, Jeong‐Yun; Irimia, A.; Loukachevitch, Lioudmila V.; Kozekov, Ivan D.; Angel, Karen C.; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter

doi:10.1074/jbc.m504756200

Cited by 139 publications

(185 citation statements)

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“…However, both of these methods are limited by the inability to resolve sequence information from a DNA mixture of variable lengths and sequences (20). Because our previous kinetic results (42) indicated Dpo4 generated a population of bypass products, these techniques could not be used.…”

Section: Resultsmentioning

confidence: 99%

Sloppy Bypass of an Abasic Lesion Catalyzed by a Y-family DNA Polymerase

Fiala

Suo

2007

Journal of Biological Chemistry

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DNA damage that eludes cellular repair pathways can arrest the replication machinery and stall the cell cycle. However, this damage can be bypassed by the Y-family DNA polymerases. Here, Dpo4, an archetypal Y-family member from the thermophilic Sulfolobus solfataricus, was used to extend our kinetic studies of the bypass of an abasic site, one of the most mutagenic and ubiquitous cellular lesions. A short oligonucleotide sequencing assay is developed to directly sequence DNA bypass products synthesized by Dpo4. Our results show that incorporation upstream of the abasic lesion is replicated error-free; yet dramatically, once Dpo4 encounters the lesion, synthesis became sloppy, with bypass products containing a myriad of mutagenic events. Incorporation of dAMP (29%) and dCMP (53%) opposite the abasic lesion at 37°C correlates exceptionally well with our kinetic results and demonstrates two dominant bypass pathways via the A-rule and the lesion loop-out mechanism. Interestingly, the percentage of overall frameshift mutations increased from 71 (37°C) to 87% (75°C). Further analysis indicates that lesion bypass via the A-rule is strongly preferred over the lesion loop-out mechanism at higher temperatures and concomitantly reduces the occurrence of "؊1 deletion" mutations observed opposite the lesion at lower temperatures. The bypass percentage via the latter pathway is confirmed by an enzymatic digestion assay, verifying the reliability of our sequencing assay. Our results demonstrate that an abasic lesion causes Dpo4 and possibly all Y-family members to switch from a normal to a very mutagenic mode of replication.Cellular DNA is continually damaged by agents both endogenous and exogenous to an organism. This damage structurally modifies DNA, arresting replication by stalling the replicative DNA polymerase. The inability to copy the genome leads to cell cycle stalling and possibly cell death. Although a large portion of DNA lesions is restored by cellular repair pathways, a biologically significant amount of damage persists. Fortunately, organisms have evolved specialized enzymes known as the Y-family DNA polymerases that are capable of replicating damaged DNA in either an error-free or error-prone fashion (1, 2), thus rescuing a stalled replisome.Although the Y-family DNA polymerases collectively synthesize one to a few nucleotides per binding event, they all lack proofreading exonuclease activity and generate up to 100,000-fold more errors on undamaged DNA when compared with the astonishingly accurate replicative DNA polymerases (1, 3, 4). This high error rate is due to the loose active sites in the structures of the eukaryotic polymerases: human polymerase (5), yeast polymerase (6), human polymerase (pol ) (7), and yeast Rev1 (8), as well as the thermophilic archaeal Sulfolobus solfataricus DNA polymerase IV (Dpo4) 3 (9) and Sulfolobus acidocaldarius Dbh (10). These more permissive active sites impart unique functional attributes to the Y-family, including the ability to replicate through aberrant and bulky DNA le...

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Section: Resultsmentioning

confidence: 99%

Sloppy Bypass of an Abasic Lesion Catalyzed by a Y-family DNA Polymerase

Fiala

Suo

2007

Journal of Biological Chemistry

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“…Wild type Dpo4 and all of the mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity as described previously (23). All unlabeled dNTPs were obtained from Amersham Biosciences, and [␥-32 P]ATP was purchased from PerkinElmer Life Sciences.…”

Section: Methodsmentioning

confidence: 99%

“…The 18-mer template strand was 5Ј-TCAC(8-oxoG)GAATCCT-TCCCCC-3Ј, and the 14-mer primer strand was 5Ј-GGGG-GAAGGATTCX-3Ј with the 3Ј-terminal nucleotide X being either A or C. The R332E(8-oxoG:C) structure was crystallized by the hanging drop vapor diffusion technique, using a mixture of 14% polyethylene glycol 4000 (w/v), 0.1 M calcium acetate, and 20 mM HEPES (pH 7.3) as reservoir. The three other mutant complexes were crystallized as described earlier (23). Only CaCl 2 was added to the protein-DNA-dGTP incubation mixture, and all buffers were Mg 2ϩ -free.…”

Section: Lc-ms/ms Analysis Of Oligonucleotide Products From Dpo4mentioning

confidence: 99%

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Hydrogen Bonding of 7,8-Dihydro-8-oxodeoxyguanosine with a Charged Residue in the Little Finger Domain Determines Miscoding Events in Sulfolobus solfataricus DNA Polymerase Dpo4

Eoff

Irimia

Angel

et al. 2007

Journal of Biological Chemistry

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“…T4 polynucleotide kinase was purchased from New England Biolabs (Beverly, MA). Dpo4 was expressed and purified as described elsewhere (51). Unmodified 13-mer, 14-mer, 24-mer, 25-mer, and 36-mer (Table 1) containing 50 mM NaCl, 5 mM dithiothreitol, 100 g of bovine serum albumin ml…”

mentioning

confidence: 99%