Sld2, Which Interacts with Dpb11 in Saccharomyces cerevisiae , Is Required for Chromosomal DNA Replication

Abstract

ABSTRACT The DPB11 gene, which genetically interacts with DNA polymerase II (ɛ), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae . To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1 ) mutations which fall into six complementation groups ( sld1 to - 6 ). In this study, we characterized SLD2 , encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1 . Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6 . The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25°C but not at 34°C when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25°C. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.