Budd Martin E, Reis Clara C, Smith Stephanie, Myung Kyungjae, Campbell Judith L
Braun Laboratories, 147-75, California Institute of Technology, Pasadena, California 91125, USA.
Mol Cell Biol. 2006 Apr;26(7):2490-500. doi: 10.1128/MCB.26.7.2490-2500.2006.
The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1delta dna2delta strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol delta, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32delta strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol delta and/or by an as yet unidentified helicase. Thus, suppression of dna2delta can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1Delta mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.
真核生物DNA复制的两个方面，冈崎片段加工（OFP）和端粒维持所需的精确机制目前还知之甚少。在这项研究中，我们提供证据表明，酿酒酵母Pif1解旋酶在DNA复制中发挥着比之前认识到的更广泛的作用，并且它可能作为OFP机制的一个组成部分与Dna2解旋酶/核酸酶协同发挥作用。此外，我们表明，已知以细胞周期特异性方式与端粒结合的Dna2可能是端粒复制装置的一个新组成部分。具体而言，我们发现缺失PIF1可抑制DNA2基因缺失突变体的致死性。pif1delta dna2delta菌株对甲磺酸甲酯仍敏感且对温度敏感；然而，通过进一步缺失pol delta的一个亚基POL32可以抑制这些表型。缺失PIF1也可抑制pol32delta菌株的冷敏感致死性和羟基脲敏感性。Dna2被认为是通过切割在OFP过程中由于pol delta和/或尚未确定的解旋酶过度的链置换而产生的长瓣来发挥作用。因此，如果缺失POL32和/或PIF1导致需要Dna2处理的长瓣减少，那么对dna2delta的抑制就可以得到合理的解释。我们进一步表明，缺失DNA2可抑制pif1Delta突变体中的长端粒表型和大量染色体重排的高形成率，这表明Dna2在没有Pif1的情况下在端粒延长中发挥作用。
