Influence of template strandedness on in vitro replication of mutagen-damaged
The authors analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo(a)pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. They found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn/sup 2 +/ and dGMP, which relax the specificity of nucleotide insertion and 3' ..-->.. 5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines, and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.
- Research Organization:
- Univ. of Chicago, IL
- OSTI ID:
- 5624700
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:9; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Cells
& Tissue Culture
560300 -- Chemicals Metabolism & Toxicology
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AROMATICS
AZAARENES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
DAYS LIVING RADIOISOTOPES
DNA
DNA POLYMERASES
DNA REPLICATION
ELECTROMAGNETIC RADIATION
ELECTROPHORESIS
ENZYMES
HETEROCYCLIC COMPOUNDS
HYBRIDIZATION
HYDROCARBONS
IN VITRO
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
NUCLEI
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYCYCLIC AROMATIC HYDROCARBONS
POLYMERASES
PURINES
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
STRAND BREAKS
TRANSFERASES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION