Other
Replication Proteins
Research on bacteriophages has been very useful in identifying replication proteins in E. coli. Some of these proteins are described below:
DNA ligase (Figure 24.24) - Covalently closes nicks in double-stranded DNA. The nick must contain 3' hydroxyl and 5' phosphoryl termini and the nucleotides being linked must be adjacent in a duplex structure and properly base-paired. DNA ligase functions to seal Okazaki fragments in the lagging strand of DNA replication.
Primase - DNA polymerases cannot initiate the synthesis of new DNA chains, but can only extend chains from preexisting 3' hydroxyl termini. Initiation of new DNA strands in E. coli begins with RNA fragments. Primase is the enzyme responsible for catalyzing synthesis of these primers. The enzyme is active only in the presence of other proteins (including a helicase), which create a complex called the primosome. DNA polymerase I and RNAse H are involved in removing RNA primers in the processing of DNA after replication. In eukaryotic cells, primase is tightly associated with DNA polymerase
(see here).
Clamps and clamp loaders - Protein
from the DNA polymerase III holoenzyme complex holds the polymerase to the DNA. This helps the DNA polymerase complex to stay on the DNA through an entire cycle of replication. A multisubunit entity called the
complex functions as the "clamp loader". That is, it loads the clamp onto the DNA. In eukaryotic cells, a multi-subunit protein called replication factor C (RF-C) is the clamp loader, and proliferating cell nuclear antigen (PCNA) is the sliding clamp.
Single-strand DNA-binding (SSB) proteins - gp32 (Figure 24.26), the most studied SSB protein, binds in a strongly cooperative fashion to single-strand DNA. That is, binding adjacent to another gp32 is much more likely than the binding of a single gp32 in isolation. This property helps promote the denaturation of duplex DNAand helps keep the DNA template in an extended, single-strand conformation, with the purine and pyrimidine bases exposed so that they can base-pair readily with incoming nucleotides. Interestingly, gp32 also promotes renaturation of single-stranded DNA. SSB proteins have been found in many organisms. In E. coli, the protein is called ssb. In eukaryotic cells, a heterotrimeric protein called replication factor A serves the role of SSB in DNA replication.
Helicases (Figure 24.28)- SSB proteins do not actively denature DNA and cannot actively unwind duplex DNA strands. Nevertheless, unwinding of this kind is essential if single-strand templates are to be exposed for polymerase action. The helicase proteins provide this function by catalyzing the ATP-dependent unwinding of double-strand DNA. E. coli contains at least 6 different helicases--some involved in DNA repair and others in conjugation. The principal helicase in DNA replication is DnaB, which interacts with DnaG and other proteins to form the primosome. All known helicases have multiple subunits. Most are homodimers, but a few are homohexamers. Figure 24.27 depicts a proposed mechanism of helicase action. In humans, two inherited diseases, Werner's syndrome and Bloom's syndrome, result from helicase defects.
Topoisomerases - Bidirectional replication of the circular E. coli chromosome unwinds about 100,000 base pairs per minute. Relief of this torsional stress is essential for DNA replication to occur. Topoisomerases are enzymes with a "swivel" mechanism that can relieve this stress. There are two general classes of topoisomerases, type I (Figure 24.30) and type II (Figure 24.31). Type I enzymes change the DNA linking number (see here for reference) in units of 1, whereas type II enzymes change the linking number in units of 2.
2. Primase Page