The eukaryotic primase/polymerase complex synthesizes approximately 10(7) primers, one per Okazaki fragment, during the replication of mammalian chromosomes, which contain 10(9) base pairs. Primase catalyzes the synthesis of a short RNA segment to a single-stranded DNA template. Primase is important in DNA replication because no known replicative DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA primer. The primase subcomplex is composed of a small catalytic subunit (p49), and a large accessory subunit (p58). Priming mechanisms remain poorly understood, although large numbers of structures of archaeal and eukaryotic p49 and/or p58 as well as structures of bacterial enzymes have been determined. In this study, we determined the structure of human p49 at 2.2 resolution with citrate in its inactive forms. Dibasic citrate was bound at the nucleotide triphosphate (NTP) beta,gamma-phosphate binding site through nine hydrogen bonds. We also measured the dissociation constant of citrate and NTPs. We further demonstrated that the p49 activity is regulated by pH and citrate, which was not previously recognized as a key regulator of DNA replication. We propose that the citrate inhibits the primase and regulates DNA replication at the replication fork. (C) 2018 Elsevier Inc. All rights reserved.