Question

 

Describe the mechanism of DNA replication in bacterial cells (like E. coli). What are the eight proteins needed for bacterial cells to make two daughter strands of DNA from one parental strand, and what is the function of each of these proteins?

Answer 1

The different proteins involved in DNA replication are:

NAME OF PROTEIN	FUNCTIONS
Dna A	Initiation of chromosome divison; binds to the origin of replicaion
Helicase	Unwinding of the double helix
Topoisomerase	introduces negative supercoils in unwinded DNA
SSB	single strand binding proteins preventing rewinding
Primase	Sythesis of RNA primers
DNA polymerase III	holoenzyme to replicate the parental strands
DNA ligase	Seals the nicks in lagging strand of DNA daughter strand

The process of replication in E.coli DNA is as follows:

The replication fork is the site of DNA replication and, by definition, includes both the DNA and associated proteins. The assembled proteins, known as the replisome, facilitate the unwinding of the helix and the addition of new nucleotides. The arrows indicate the direction of movement of the replication fork.
The synthesis of two DNA helices results from adding a new complementary strand to each one of the separated old strands.For the replication fork to proceed, both the double helix and the positive
supercoils must be unwound. Helicase unwinds the double helix and DNA gyrase (a type II topoisomerase) removes the positive supercoiling. To unwind DNA, helicase first binds to DNA and then causes the dissociation of hydrogen bonds connecting base pairs to separate the strands of the helix. This process requires energy input from ATP hydrolysis. Soon after DNA helicase breaks the hydrogen bonds holding the DNA strands together, SSB protein binds to the freed strands to keep them from re-annealing and also to prevent
intra-strand pairing.The protein at the replication fork responsible for DNA synthesis, the DNA polymerase, always synthesizes DNA in the 5'- to 3'-direction. Therefore one new strand (leading strand) can be made continuously, while the other (lagging strand) must be made discontinuously (i.e., in short segments).To begin a new strand DNA polymerase uses a short RNA primer made by another enzyme. Unlike DNA polymerases, RNA polymerases can start new strands. A special RNA polymerase, known as primase (DnaG protein) makes the RNA primers that are responsible for strand initiation during DNA synthesis in bacteria. Although the leading strand only
needs to be started once, the lagging strand is made in short sections and a new RNA primer must be inserted each time a new portion is made. Thus DNA polymerase will build new strands of DNA starting from each RNA primer. Each time a new Okazaki fragment is begun, the Pol III assembly that is making the
lagging strand releases its grip on the DNA and relocates to start making a new strand of DNA starting from the 3‘-end of the RNA primer. After the replication fork has passed by, the lagging strand is left as a series of Okazaki
fragments with gaps (that is, spaces from which one or more nucleotides are missing) between them. The gaps are filled with the RNA primers. Joining the Okazaki fragments to give a complete strand of DNA is accomplished by
two—or perhaps three—enzymes working in succession: Ribonuclease H (RNase H), DNA polymerase I (Pol I), and DNA ligase. DNA polymerase I both degrades the RNA primers (by 5'→3'exonuclease acitivity) and fills the gaps left by the degraded RNA. Finally, DNA ligase joins the ends.