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Translesion DNA synthesis by human DNA polymerase eta on templates containing a pyrimidopurinone deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one.

Stafford JB, Eoff RL, Kozekova A, Rizzo CJ, Guengerich FP, Marnett LJ - Biochemistry (2009)

Bottom Line: M(1)dG partially blocked DNA synthesis by polymerase eta.Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M(1)dG lesion (800-fold less efficient than opposite dG).Extension from M(1)dG.dC was equally as efficient as from control primer-templates (dG.dC). dATP insertion opposite M(1)dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M(1)dG.dA was 20-fold less efficient than dG.dC.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

ABSTRACT
M(1)dG (3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M(1)dG to dT and M(1)dG to dA) and frameshift mutations. M(1)dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M(1)dG in Escherichia coli is dependent on the SOS response, specifically the umuC and umuD gene products, suggesting that mutagenic lesion bypass occurs by the action of translesion DNA polymerases, like DNA polymerase V. Bypass of DNA lesions by translesion DNA polymerases is conserved in bacteria, yeast, and mammalian cells. The ability of recombinant human DNA polymerase eta to synthesize DNA across from M(1)dG was studied. M(1)dG partially blocked DNA synthesis by polymerase eta. Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M(1)dG lesion (800-fold less efficient than opposite dG). Extension from M(1)dG.dC was equally as efficient as from control primer-templates (dG.dC). dATP insertion opposite M(1)dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M(1)dG.dA was 20-fold less efficient than dG.dC. The sequences of full-length human DNA polymerase eta bypass products of M(1)dG were determined by LC-ESI/MS/MS. Bypass products contained incorporation of dA (52%) or dC (16%) opposite M(1)dG or -1 frameshifts at the lesion site (31%). Human DNA polymerase eta bypass may lead to M(1)dG to dT and frameshift but likely not M(1)dG to dA mutations during DNA replication.

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LC-ESI/MS/MS analysis of products from the in vitro replication of M1dG containing DNA templates by human DNA polymerase η. (A) Total ion current trace. (B) Total mass spectrum of the mixture of M1dG bypass products. (C) CID spectrum of m/z 1098.7 with sequence TCAGTGA. (D) CID spectrum of m/z 1087.2 with sequence TCCGTGA. (E) CID spectrum of m/z 942.2 with sequence TC∧GTGA. (F) CID spectrum of m/z 836.5 with sequence TCAGTGAA. DNA bases shown in bold italic were incorporated opposite M1dG lesions in the template DNA.
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fig5: LC-ESI/MS/MS analysis of products from the in vitro replication of M1dG containing DNA templates by human DNA polymerase η. (A) Total ion current trace. (B) Total mass spectrum of the mixture of M1dG bypass products. (C) CID spectrum of m/z 1098.7 with sequence TCAGTGA. (D) CID spectrum of m/z 1087.2 with sequence TCCGTGA. (E) CID spectrum of m/z 942.2 with sequence TC∧GTGA. (F) CID spectrum of m/z 836.5 with sequence TCAGTGAA. DNA bases shown in bold italic were incorporated opposite M1dG lesions in the template DNA.

Mentions: The substitution of M1dG for dG resulted in four major products. The major ions observed in the total mass spectrum resulted from one product with m/z of 1098.7 (doubly charged ion) and 732.2 (triply charged ion), a second with m/z of 1087.2 (doubly charged ion) and 724.5 (triply charged ion), a third with m/z 942.2 (doubly charged ion), and a fourth with m/z of 836.5 (doubly charged ion) (Figure 5B). Sequence assignments were made based on CID fragmentation patterns. The strong signal-to-noise ratio of the CID spectra indicated that the in vitro polymerase reaction had proceeded with good yield. The major product (m/z of 1098.7 and 732.2) corresponded to the incorporation of dA opposite M1dG (5′-pTCAGTGA-3′, Figure 5C and Table S2). Incorporation of dC opposite M1dG (m/z 1087.2 and 732.2, 5′-pTCCGTGA-3′) was also observed and had a nearly identical CID fragmentation pattern as observed for the major product of control reactions (Figure 5D and Table S2). Minor products were also observed and included incorporation of dA opposite M1dG with “blunt-end” addition of dA (m/z 836.5, Figure 5F and Table S2) or dG (m/z 1263.2, CID data not shown). Incorporation of dT or dG opposite M1dG was not observed.


Translesion DNA synthesis by human DNA polymerase eta on templates containing a pyrimidopurinone deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one.

Stafford JB, Eoff RL, Kozekova A, Rizzo CJ, Guengerich FP, Marnett LJ - Biochemistry (2009)

LC-ESI/MS/MS analysis of products from the in vitro replication of M1dG containing DNA templates by human DNA polymerase η. (A) Total ion current trace. (B) Total mass spectrum of the mixture of M1dG bypass products. (C) CID spectrum of m/z 1098.7 with sequence TCAGTGA. (D) CID spectrum of m/z 1087.2 with sequence TCCGTGA. (E) CID spectrum of m/z 942.2 with sequence TC∧GTGA. (F) CID spectrum of m/z 836.5 with sequence TCAGTGAA. DNA bases shown in bold italic were incorporated opposite M1dG lesions in the template DNA.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651650&req=5

fig5: LC-ESI/MS/MS analysis of products from the in vitro replication of M1dG containing DNA templates by human DNA polymerase η. (A) Total ion current trace. (B) Total mass spectrum of the mixture of M1dG bypass products. (C) CID spectrum of m/z 1098.7 with sequence TCAGTGA. (D) CID spectrum of m/z 1087.2 with sequence TCCGTGA. (E) CID spectrum of m/z 942.2 with sequence TC∧GTGA. (F) CID spectrum of m/z 836.5 with sequence TCAGTGAA. DNA bases shown in bold italic were incorporated opposite M1dG lesions in the template DNA.
Mentions: The substitution of M1dG for dG resulted in four major products. The major ions observed in the total mass spectrum resulted from one product with m/z of 1098.7 (doubly charged ion) and 732.2 (triply charged ion), a second with m/z of 1087.2 (doubly charged ion) and 724.5 (triply charged ion), a third with m/z 942.2 (doubly charged ion), and a fourth with m/z of 836.5 (doubly charged ion) (Figure 5B). Sequence assignments were made based on CID fragmentation patterns. The strong signal-to-noise ratio of the CID spectra indicated that the in vitro polymerase reaction had proceeded with good yield. The major product (m/z of 1098.7 and 732.2) corresponded to the incorporation of dA opposite M1dG (5′-pTCAGTGA-3′, Figure 5C and Table S2). Incorporation of dC opposite M1dG (m/z 1087.2 and 732.2, 5′-pTCCGTGA-3′) was also observed and had a nearly identical CID fragmentation pattern as observed for the major product of control reactions (Figure 5D and Table S2). Minor products were also observed and included incorporation of dA opposite M1dG with “blunt-end” addition of dA (m/z 836.5, Figure 5F and Table S2) or dG (m/z 1263.2, CID data not shown). Incorporation of dT or dG opposite M1dG was not observed.

Bottom Line: M(1)dG partially blocked DNA synthesis by polymerase eta.Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M(1)dG lesion (800-fold less efficient than opposite dG).Extension from M(1)dG.dC was equally as efficient as from control primer-templates (dG.dC). dATP insertion opposite M(1)dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M(1)dG.dA was 20-fold less efficient than dG.dC.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

ABSTRACT
M(1)dG (3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M(1)dG to dT and M(1)dG to dA) and frameshift mutations. M(1)dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M(1)dG in Escherichia coli is dependent on the SOS response, specifically the umuC and umuD gene products, suggesting that mutagenic lesion bypass occurs by the action of translesion DNA polymerases, like DNA polymerase V. Bypass of DNA lesions by translesion DNA polymerases is conserved in bacteria, yeast, and mammalian cells. The ability of recombinant human DNA polymerase eta to synthesize DNA across from M(1)dG was studied. M(1)dG partially blocked DNA synthesis by polymerase eta. Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M(1)dG lesion (800-fold less efficient than opposite dG). Extension from M(1)dG.dC was equally as efficient as from control primer-templates (dG.dC). dATP insertion opposite M(1)dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M(1)dG.dA was 20-fold less efficient than dG.dC. The sequences of full-length human DNA polymerase eta bypass products of M(1)dG were determined by LC-ESI/MS/MS. Bypass products contained incorporation of dA (52%) or dC (16%) opposite M(1)dG or -1 frameshifts at the lesion site (31%). Human DNA polymerase eta bypass may lead to M(1)dG to dT and frameshift but likely not M(1)dG to dA mutations during DNA replication.

Show MeSH
Related in: MedlinePlus