Molecular cloning and characterization of a Streptococcus sanguis DNase necessary for repair of DNA damage induced by UV light and methyl methanesulfonate

Journal Article · Wed Jul 01 00:00:00 EDT 1987 · J. Bacteriol.; (United States)

OSTI ID:6170079

Lindler, L E; Macrina, F L

We developed a method for cloning cellular nucleases from streptococci. Recombinant lambda gt11 bacteriophage containing streptococcal nuclease determinants were identified by the production of pink plaques on toluidine blue O DNase plates. We used this technique to clone a 3.2-kilobase-pair EcoRI fragment with DNase activity from the chromosome of Streptococcus sanguis. The locus was designated don (DNase one) and could be subcloned and stably maintained on plasmid vectors in Escherichia coli. Minicell analyses of various subclones of the don locus allowed us to determine the coding region and size of the Don nuclease in E. coli. The don gene product had an apparent molecular mass of 34 kilodaltons and degraded native DNA most efficiently, with lesser activity against denatured DNA and no detectable activity against RNA. S. sanguis don deletion mutants were constructed by transformation of competent cells with in vitro-prepared plasmid constructs. S. sanguis don deletion mutants retained normal transformation frequencies for exogenously added donor DNA. However, when compared with Don+ wild-type cells, these mutants were hypersensitive to DNA damage induced by UV light and methyl methanesulfonate. An S. sanguis don-specific DNA probe detected homology to chromosomal DNA isolated from Streptococcus pneumoniae and Streptococcus mutans Bratthall serogroups d and g. Our results suggested that the don locus was the S. sanguis allele of the previously described S. pneumoniae major exonuclease and was involved in repair of DNA damage. Furthermore, hybridization studies suggested that the don locus was conserved among species of oral streptococci.

• Research Organization: Virginia Commonwealth Univ., Richmond
• OSTI ID: 6170079
• Journal Information: J. Bacteriol.; (United States), Vol. 169:7
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
DNA-ASE
CHEMICAL COMPOSITION
CLONING
METHYL METHANESULFONATE
GENETIC EFFECTS
STREPTOCOCCUS
DNA REPAIR
GENETIC RADIATION EFFECTS
BACTERIOPHAGES
CHROMOSOMES
ENZYME ACTIVITY
ESCHERICHIA COLI
HYBRIDIZATION
ULTRAVIOLET RADIATION
BACTERIA
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
ELECTROMAGNETIC RADIATION
ENZYMES
ESTERASES
ESTERS
HYDROLASES
MICROORGANISMS
MUTAGENS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
PHOSPHODIESTERASES
RADIATION EFFECTS
RADIATIONS
RECOVERY
REPAIR
SULFONIC ACID ESTERS
VIRUSES
560130* - Radiation Effects on Microorganisms