Umezu K, Sugawara N, Chen C, Haber J E, Kolodner R D
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Genetics. 1998 Mar;148(3):989-1005. doi: 10.1093/genetics/148.3.989.
Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.
复制蛋白A（RPA）是一种单链DNA结合蛋白，被鉴定为体外SV40 DNA复制的必需因子。为了了解RPA在体内的功能，我们对酿酒酵母RFA1基因进行了诱变，鉴定出19个对紫外线（UV）照射和甲基磺酸甲酯（MMS）敏感的突变体以及5个温度敏感突变体。对UV和MMS敏感的突变体对这些试剂的敏感性增加了10^4到10^5倍。一些对UV和MMS敏感的突变体在MAT处被HO诱导的双链断裂杀死。对一个对UV和MMS敏感的rfa1突变体中的重组进行物理分析表明，它在交配型转换和单链退火重组方面存在缺陷。对两个温度敏感突变体进行了详细表征，发现在限制温度下它们具有停滞表型和表明DNA复制不完全的DNA含量。DNA序列分析表明，大多数突变改变了酵母、人类和非洲爪蟾RPA1之间保守的氨基酸。综上所述，我们得出结论，RPA1在体内具有多种作用，在DNA复制、修复和重组中发挥功能，类似于细菌和噬菌体的单链DNA结合蛋白。
