The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase ␦ has not been clearly defined. We show that the third subunit of human DNA polymerase ␦, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase ␦ activity and for binding of the cell cycle inhibitor p21Cip1 . In accordance with this, a peptide containing the PCNA-binding domain of p21Cip1 inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase ␦. Furthermore, pull-down assays showed that DNA polymerase ␦ requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase ␦ displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21 Cip1 . Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase ␦ within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase ␦.DNA replication requires the recruitment of multiple components during the S phase of the cell cycle. Of chief importance are DNA polymerases, of which three, ␣, ␦, and ⑀, have been shown by genetic studies in the yeast Saccharomyces cerevisiae to be essential for the replication of the eucaryotic genome (1, 2). Among these, DNA polymerase ␣-primase is the only enzyme that can start DNA synthesis de novo. It is thus ideally suited for the synthesis of short primers that serve to initiate leading strand synthesis at the replication origin and Okazaki fragments on the lagging strand (3, 4). Both DNA polymerases ␦ and ⑀ can elongate primers synthesized by DNA polymerase ␣ and appear to have overlapping or complementary functions (5). Although DNA polymerase ␦ alone can replicate both leading and lagging strands in vitro (5), mounting evidence suggests that DNA polymerase ⑀ may be implicated in DNA replication in ways that may not necessarily involve its DNA polymerase activity (6 -10).We have been interested in how DNA polymerase ␦ interacts with other factors to accomplish replication of the mammalian genome. Extensive research over a number of years has shown that DNA polymerase ␦ requires interactions with at least two factors, a toroidal-shaped homotrimeric protein known as proliferating cell nuclear antigen (PCNA) 1 and a heteropentameric complex termed replication factor C. In the presence of ATP, replication factor C interacts with the 3Ј primer-template terminus and recruits PCNA onto the DNA, creating a mobile platform that tethers DNA polymerase ␦ to the primer terminus. Once bound to PCNA, DNA polymerase ␦ acquires new dynamic properties, including high processivity, low nonpro...

Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication.

Mycolic acids (MAs) have a strategic location within the mycobacterial envelope, deeply influencing its architecture and permeability, and play a determinant role in the pathogenicity of mycobacteria. The fatty acid synthase type II (FAS-II) multienzyme system is involved in their biosynthesis. A combination of pull-downs and proteomics analyses led to the discovery of a mycobacterial protein, HadD, displaying highly specific interactions with the dehydratase HadAB of FAS-II. In vitro activity assays and homology modeling showed that HadD is, like HadAB, a hot dog folded (R)-specific hydratase/dehydratase. A hadD knockout mutant of Mycobacterium smegmatis produced only the medium-size alpha’-MAs. Data strongly suggest that HadD is involved in building the third meromycolic segment during the late FAS-II elongation cycles, leading to the synthesis of the full-size alpha- and epoxy-MAs. The change in the envelope composition induced by hadD inactivation strongly altered the bacterial fitness and capacities to aggregate, assemble into colonies or biofilms and spread by sliding motility, and conferred a hypersensitivity to the firstline antimycobacterial drug rifampicin. This showed that the cell surface properties and the envelope integrity were greatly affected. With the alarmingly increasing case number of nontuberculous mycobacterial diseases, HadD appears as an attractive target for drug development.