Template length, sequence context, and 3 prime -5 prime exonuclease activity modulate replicative bypass of thymine glycol lesions in vitro
- Yale Univ. School of Medicine, New Haven, CT (USA)
cis-Thymine glycol, a product of ionizing radiation damage to DNA, has been introduced quantitatively at a single site into oligonucleotide templates. The ability of DNA polymerases to replicate templates containing thymine glycol was studied by a primer extension assay, and three factors that influence replicative bypass of this lesion in vitro have been identified. These factors include template length, sequence context, and 3{prime}-5{prime} exonuclease activity. Synthesis by the large fragment of DNA polymerase I (Klenow fragment) terminates quantitatively opposite thymine glycol when the template strand extends only two nucleotides beyond the lesion. Significant bypass is observed when the length of the template beyond the lesion is increased to six nucleotides. On the longer templates, the frequency of bypass of the Klenow fragment depends upon the identity of the base immediately 5{prime} to thymine glycol. The extent of bypass is greatest with cytosine and least with adenine at this position. Bypass of thymine glycol lesions by DNA polymerase {alpha}{sub 2} from HeLa cells shows a qualitatively similar dependence upon local sequence context. In contrast, synthesis by T4 DNA polymerase is quantitatively blocked opposite the lesion regardless of template length or DNA sequence context. Synthesis by a mutant Klenow fragment that is deficient in 3{prime}-5{prime} exonuclease activity, or by AMV reverse transcriptase, results in a significant increase in the frequency of bypass. Thus, increased nucleotide turnover at, or beyond, the site of the lesion is likely to contribute significantly to the arrest of synthesis provoked by cis-thymine glycol in vitro.
- OSTI ID:
- 5560996
- Journal Information:
- Biochemistry; (USA), Vol. 28:2; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
Similar Records
A Crystallographic Study of the Role of Sequence Context in Thymine Glycol Bypass by a Replicative DNA Polymerase Serendipitously Sheds Light on the Exonuclease Complex
Synthesis and structure-activity relationships of site-specific cis-syn and trans-syn thymine dimers
Related Subjects
DNA
BIOLOGICAL RADIATION EFFECTS
DNA REPAIR
BIOLOGICAL PATHWAYS
AUTORADIOGRAPHY
DNA POLYMERASES
DNA SEQUENCING
ENZYME ACTIVITY
GLYCOLS
OLIGONUCLEOTIDES
PHOSPHORUS 32
THYMINE
ALCOHOLS
AZINES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
DAYS LIVING RADIOISOTOPES
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PYRIMIDINES
RADIATION EFFECTS
RADIOISOTOPES
RECOVERY
REPAIR
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
URACILS
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture