Okazaki fragment maturation is an important process in eukaryotic DNA replication for maintaining genome integrity. The principal mechanism is processing generation of 5’-flaps which have the potential to form a variety of structure. Obviously, accumulation of unprocessed flaps is detrimental to cells, which potentially leads to mutations and chromosome aberrations that ultimately cause diseases. Dna2 and Rad27 (yeast Fen1; Flap-endonuclease 1) play central role in removing the unprocessed flaps that arise during DNA lagging strand replication process. In previous research, genetic interaction between Rad27 and HCS1 (yeast DNA helicase A; DNA polymerase α- associated helicase A), and possible involvements of HCS1 in the lagging strand synthesis were reported. We aimed to elucidate the relevance of HCS1 in flap processing with Dna2 and Rad27. We showed that the overexpression of HCS1 can suppress the lethality of Dna2 helicase mutants in Saccharomyces cerevisiae, which indicates the involvement of HCS1 in flap processing in vivo. It was also found that HCS1 strongly interacts with Rad27 and PCNA in vitro. Nuclease assay showed HCS1 stimulate the nuclease activities of Rad27 and Dna2.