Somatic mutation rates and specificities at TC/AG and GT/CA microsatellite sequences in nontumorigenic human lymphoblastoid cells.

We have examined mutational events at TC/AG microsatellites, the second most abundant dinucleotide repetitive motif in the human genome. Mutational targets were constructed containing TC/AG alleles up to 20 units in-frame within the coding region of the herpes simplex virus thymidine kinase (HSV-tk) gene. These targets were incorporated into oriP shuttle vectors, which replicate episomally in human lymphoblastoid cells. The overall HSV-tk mutant frequencies measured after 10 population doublings in cells derived from a clinically normal donor were slightly increased over the background of mutations recovered in Escherichia coli. DNA sequence analyses revealed that replication of TC/AG vectors in human cells increased the mutation frequencies at the microsatellite motif up to 3-fold, relative to background. Additionally, the median HSV-tk mutation rate of single-cell clones carrying the [TC/AG]17 vector was significantly different from that of clones harboring the control vector. The median rate of allele length alterations within the [TC/AG]11 tract was 2 x 10(-6) mutations/cell generation, with an equivalent rate of deletion and expansion mutations. In contrast, a [GT/CA]10 vector showed no increase in microsatellite mutation frequency after replication in human cells, and mutation rates of clones carrying a [GT/CA]16 vector were not significantly different from controls. Intriguingly, replication in human cells of all microsatellite-containing vectors resulted in elevated mutation frequencies at the downstream HSV-tk coding sequence of up to 20-fold, an effect not observed for the control vector. These results demonstrate that the frequency of mutational events at TC/AG motifs is slightly greater than at GT/CA motifs of similar allele length. This is the first report to our knowledge of the mutation rates at TC/AG microsatellite alleles in eukaryotic or prokaryotic cells.

Alkylation-induced frameshift mutagenesis during in vitro DNA synthesis by DNA polymerases alpha and beta.

We have analyzed the mutational spectra produced during in vitro DNA synthesis by DNA polymerase alpha-primase and DNA polymerase beta. The polymerase mutation frequency as measured in the in vitro herpes simplex virus thymidine kinase (HSV-tk) forward assay was increased when reactions utilized single-stranded DNA templates randomly modified by 20 mM N-ethyl-N-nitrosourea (ENU), relative to solvent-treated templates. A 20- to 50-fold increase in the frequency of G-->A transition mutations was observed for both polymerases, as expected due to mispairing by O6-ethylguanine lesions. Strikingly, ENU treatment of the template also resulted in a five- to 12-fold increased frequency of frameshift errors at heteropolymeric (non-repetitive) template sequences produced by polymerase beta and polymerase alpha-primase, respectively. The increased proportion of frameshift mutations at heteropolymeric sequences relative to homopolymeric (repetitive) sequences produced by each polymerase in response to ENU damage was statistically significant. For polymerase alpha-primase, one-base deletion errors at template guanine residues was the second most frequent mutational event, observed at a frequency only four-fold lower than the G-->A transition frequency. In the polymerase beta reactions, the frequency of insertion errors at homopolymeric (repetitive) sequences was increased six-fold using alkylated templates, relative to solvent controls. The frequency of such insertion errors was only three-fold lower than the frequency of G-->A transition errors by polymerase beta. Although ENU is generally regarded as a potent base substitution mutagen, these data show that monofunctional alkylating agents are capable of inducing frameshift mutations in vitro. Alkylation-induced frameshift mutations occur in both repetitive and non-repetitive DNA sequences; however, the mutational specificity is dependent upon the DNA polymerase.

Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions.

We have developed an in vitro DNA polymerase forward mutation assay using damaged DNA templates that contain the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. The quantitative method uses complementary strand hybridization to gapped duplex DNA molecules and chloramphenicol selection. This design ensures exclusive analysis of mutations derived from the DNA strand produced during in vitro synthesis. We have examined the accuracy of DNA synthesis catalyzed by calf thymus polymerase alpha-primase, polymerase beta and exonuclease-deficient Klenow polymerase. Using unmodified DNA templates, polymerase beta displays a unique specificity for the loss of two bases in a dinucleotide repeat sequence within the HSV-tk locus. Treatment of the DNA template with N-ethyl-N-nitrosourea resulted in a dose-dependent inhibition of DNA synthesis concomitant with an increased mutation frequency. Similar dose-response curves were measured for the three polymerases examined; thus the identity of the DNA polymerase does not appear to affect the mutagenic potency of ethyl lesions. The HSV-tk system is unique in that damage-induced mutagenesis can be analyzed both quantitatively and qualitatively in human cells, in bacterial cells and in in vitro DNA synthesis reactions at a single target sequence.