DNA cytosine methylation and DNA repair
Description
Thymine$\cdot$guanine (T$\cdot$G) and uracil$\cdot$guanine (U$\cdot$G) mismatched base pairs in DNA are formed by the spontaneous deamination of 5-methylcytosine (m$\sp5$C) and cytosine (C) residues, respectively. Covalently closed circular (CCC) heteroduplex M13mp18 replicative form (RF) DNAs with a T$\cdot$G or a U$\cdot$G mismatch in a unique restriction site were created by oligonucleotide-directed mutagenesis and were transfected into repair-proficient and repair-deficient E. coli. The CCC heteroduplex M13mp18 DNAs were unmethylated at all dam (5$\sp\prime$-GATC-3$\sp\prime$) sites in order to avoid methylation-directed and nick-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase ($ung\sp{+}$) and which is proficient in mismatch repair ($mut\sp{+}$), $\sim$97% of the U$\cdot$G-containing heteroduplex had the U residue excised by the uracil excision repair system. With the analogous T$\cdot$G mispair, mismatch repair operated on almost all the transfecting heteroduplexes to remove the T residue in 75% of the heteroduplexes when the T residue was on the minus-strand of the M13mp18 RF DNA. Similar preferential removal of the minus-strand's mismatched base was observed. In $ung\sp{-}$ hosts, which lack uracil-DNA glycosylase but have an intact Mut system, the U$\cdot$G mismatches are corrected by mismatch repair with the same directionality and to the same extent as were T$\cdot$G mispairs in $ung\sp{-}$ or $ung\sp{+}$ hosts. In E. coli hosts carrying insertions, deletions, or point mutations in the genes coding for the various components of the Mut system (mutL, mutS, MutH genes), the mismatch repair of T$\cdot$G mismatches is diminished but not abolished The ability of restriction endonucleases to hydrolyze their DNA recognition sites substituted with mismatched base pairs was investigated. The restriction enzymes studied included SalI, AccI, HincII, HindIII, SmaI, SstI, and KpnI. None of the restriction enzymes were able to completely cleave the mismatch-containing recognition sites under standard conditions. However, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatch-containing recognition site. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs. Also this study yielded information on how different pyrimidine functional groups interacted with the restriction enzymes. (Abstract shortened with permission of author.)