No abstract

DNA polymerase is the critical enzyme maintaining genetic integrity during DNA replication. Individual steps in the replication process that contribute to DNA synthesis fidelity include nucleotide insertion, exonucleolytic proofreading, and binding to and elongation of matched and mismatched primer termini. Each process has been investigated using polyacrylamide gel electrophoresis (PAGE) to resolve 32P-labeled primer molecules extended by polymerase. We describe how integrated gel band intensities can be used to obtain site-specific velocities for addition of correct and incorrect nucleotides, extending mismatched compared to correctly matched primer termini and measuring polymerase dissociation rates and equilibrium DNA binding constants. The analysis is based on steady-state "single completed hit conditions", where polymerases encounter many DNA molecules but where each DNA encounters an enzyme at most once. Specific topics addressed include nucleotide misinsertion, mismatch extension, exonucleolytic proofreading, single nucleotide discrimination using PCR, promiscuous mismatch extension by HIV-1 and AMV reverse transcriptases, sequence context effects on fidelity and polymerase dissociation, structural and kinetic properties of mispairs relating to fidelity, error avoidance mechanisms, kinetics of copying template lesions, the "A-rule" for insertion at abasic template lesions, an interesting exception to the "A-rule", thermodynamic and kinetic determinants of base pair discrimination by polymerases.

Ovarian failure leading to infertility can be caused by improper prenatal development of the fetal gonad or disruption of the complex postnatal process of folliculogenesis due to alterations in intragonadal or extragonadal regulation. It is critical to have physiological models that mimic events occurring during human development to understand, treat, and prevent ovarian failure in women. Many workers have chosen the mouse as the mammalian model with which to study ovarian function. This review summarizes several key events in female gonadogenesis and folliculogenesis in mice with specific emphasis on spontaneous or induced mutations yielding mouse models that have female infertility owing to ovarian failure. 184 J. A. Elvin and M. M. Matzuk Cohen P, Zhu L and Pollard J (1997) Absence of colony stimulating factor-1 in osteopetrotic (csfm op /csfm op) mice disrupts estrous cycles and ovulation Biology of Reproduction 56 110-118 *Colledge W, Carlton M, Udy G and Evans M (1994) Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs Nature 370 65-68 Coulombre J and Russell E (1954) Analysis of the pleiotropism at the W-locus in the mouse: the effects of W and W v substitution upon postnatal development of germ cells Journal of Experimental Zoology 126 277-296 Couse J, Curtis S, Washburn T, Lindzey J, Golding T, Lubahn D, Smithies O and Korach K (1995) Analysis of transcription and oestrogen insensitivity in the female mouse after targeted disruption of the oestro-gen receptor gene Molecular Endocrinology 9 1441-1454 Dinchuk J, Car B, Focht R, Johnston J, Jaffee B, Covington M, Contel N, Eng V, Collins R, Czerniak P, Gorry S and Trzaskos J (1995) Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II Nature 378 406-409 *Dong J, Albertini D, Nishimori K, Kumar T, Lu N and Matzuk M (1996) Growth differentiation factor-9 is required during early ovarian folliculo-genesis Nature 383 531-535

Mice carrying a null mutation in the mismatch repair gene Msh6 were generated by gene targeting. Cells that were homozygous for the mutation did not produce any detectable MSH6 protein, and extracts prepared from these cells were defective for repair of single nucleotide mismatches. Repair of 1, 2, and 4 nucleotide insertion/deletion mismatches was unaffected. Mice that were homozygous for the mutation had a reduced life span. The mice developed a spectrum of tumors, the most predominant of which were gastrointestinal tumors and B- as well as T-cell lymphomas. The tumors did not show any microsatellite instability. We conclude that MSH6 mutations, like those in some other members of the family of mismatch repair genes, lead to cancer susceptibility, and germline mutations in this gene may be associated with a cancer predisposition syndrome that does not show microsatellite instability.