Architecture and function of the phage T4 replication fork

In the phageT4 replication system, T4 DNA polymerase is held on both strands by the gene 45 clamp, loaded by the 44/62 clamp loader. 41 Helicase, loaded by 59 protein, opens the duplex and enables RNA primer synthesis by 61 primase. 32 Protein coats the lagging strand template. 59 Protein helps to coordinate synthesis of the two strands at the fork by blocking leading strand synthesis until 32 protein is loaded. Interaction between T4 5′ nuclease (RNaseH) and 32 protein is required for efficient maturation of lagging strand fragments. In our crystal structure of T4 RNaseH with a short fork substrate, the 3′ arm, corresponding to the lagging strand template, reaches close to the binding site for 32 protein at the C‐terminus of the nuclease. Electron microscopy shows a single complex of the leading and lagging strand proteins at the fork, with a “trombone” loop present on more than 50% of the molecules. The protein covered ssDNA on the lagging strand is folded into highly compact structures. Using DNA “pointers” to biotin‐tagged proteins, we find the expected two polymerases on many of the complexes. 59 Protein remains on the fork after loading the helicase. Helicase is present on a large fraction of actively replicating molecules, but on a smaller fraction of molecules with a stalled polymerase. The T4 system can continue both leading and lagging strand synthesis, when a limited number of lagging strand fragments are terminated. Supported by the Intramural Research Program, NIDDK, NIH (NGN), National Institutes of Health (JDG), and The National Science Foundation (TCM).