Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin
situations, an RNA primer for DNA replication can be made by the RNA polymerase used for information processing (i.e., the RNA polymerase that makes all the other RNAs, including mRNA, tRNA, and rRNA; see chapter 2). Unlike DNA polymerase, primase and RNA polymerases do not require a primer to initiate the synthesis of new strands. During DNA replication, a special enzyme activity recognizes and removes the RNA primer (see below).
Table 1.1 Proteins involved in Escherichia coli DNA replication
| Protein | Gene | Function |
|---|---|---|
| DnaA | dnaA | Initiator protein; primosome (priming complex) formation |
| DnaB | dnaB | DNA helicase |
| DnaC | dnaC | Delivers DnaB to replication complex |
| SSB | ssb | Binding to single-stranded DNA |
| Primase | dnaG | RNA primer synthesis |
| DNA ligase | lig | Sealing DNA nicks |
| DNA gyrase | Supercoiling | |
| α | gyrA | Nick closing |
| β | gyrB | ATPase |
| DNA Pol I | polA | Primer removal; gap filling |
| DNA Pol III (holoenzyme) | ||
| α | dnaE | Polymerization |
| ε | dnaQ | 3′-to-5′ editing |
| RNase H | rnhA | Can aid in RNA primer removal |
| θ | holE | Present in core (αεθ) |
| β | dnaN | Sliding clamp |
| τa | dnaX | Organizes complex; joins leading and lagging DNA Pol III |
| γb | dnaX | Binds clamp loaders and single-strand-binding protein |
| δ | holA | Clamp loading |
| δ' | holB | Clamp loading |
| χ | holC | Binds single-strand-binding protein |
| φ | holD | Holds χ to the clamp loader |
aFull-length product of the dnaX gene.
bShorter product of the dnaX gene produced by translational frameshifting (see chapter 2).
NUCLEASES
Enzymes that degrade DNA strands by breaking the phosphodiester bonds are just as important in replication as the enzymes that polymerize DNA by forming phosphodiester bonds between the nucleotides. These bondbreaking enzymes, called nucleases, can be grouped into two major categories. One type can initiate breaks in the middle of a DNA strand and so are called endonucleases, from a Greek word meaning “within,” and the other type can remove nucleotides only from the ends of DNA strands and so are called exonucleases, from a Greek word meaning “outside.” A special type of endonuclease activity, called a flap endonuclease activity, is involved in primer removal by DNA polymerase I. The flap endonuclease activity appears to be common to all organisms for removing RNA primers. In E. coli, DNA polymerase I displaces the RNA primer, making a flap-like structure, and then the flap endonuclease activity of Pol I cleaves away the oligonucleotide as indicated (Figure 1.9). The exonucleases can be subdivided into two groups. Some exonucleases can degrade only from the 3′ end of a DNA strand, degrading DNA in the 3′-to-5′ direction. These are called 3′ exonucleases; one example of their activity is their role in the editing function associated with DNA polymerases I and III, which is discussed below. Other exonucleases, called 5′ exonucleases, degrade DNA strands only from the 5′ end.
DNA LIGASES
DNA ligases are enzymes that form phosphodiester bonds between the ends of separate presynthesized chains of DNA. This important function cannot be performed by any of the known DNA polymerases. During replication, ligase joins the 5′ phosphate at the end of one DNA chain to the 3′ hydroxyl at the end of another chain to make a longer, continuous chain (Figure 1.8).
ACCESSORY PROTEINS
Replication of large DNAs requires many functions that reside in proteins separate from the subunit used for polymerizing the chain of nucleotides. These functions include the coordination of multiple DNA polymerases and tethering of these components to the template DNA strands as a moving production platform. DNA polymerase III is the major DNA replication protein in E. coli responsible for polymerizing the new complementary DNA strands, and it functions with multiple DNA polymerase accessory proteins that travel along the template strand with the molecule of DNA polymerase III. The term DNA polymerase III holoenzyme can be used to describe the entire complex of proteins. The various subunits and subassemblies of the DNA polymerase III holoenzyme were originally identified from fractionation procedures and were designated by Greek letters (Table 1.1).