Module 7 : Concept of GENE, GENOME and GENE EXPRESSION

Enzymes involved in DNA replication.

Enzymes involved :

DNA-dependent DNA polymerase :

• First discovered in 1958 by Arthur Kornberg; Later, DNA-pol II and DNA-pol III were identified in experiments using mutant E.coli cell line.

Structure of DNA polymerase III :

Klenow fragment :

The Klenow fragment is a large protein fragment produced when prokaryotic DNA polymerase I is cleaved by the protease subtilisin. Two fragments ( one large and one small) are generated as a result of proteolysis : • Large fragment is called Klenow fragment. It possess 5´ --> 3' DNA polymerization activity and 3´ --> 5´exonuclease activity for proofreading, but 5' --> 3' exonuclease activity is lost. • Small fragment retains 5´- --> 3´ exonuclease activity but does not have the other two activities exhibited by the Klenow fragment (i.e. 5'- --> 3' polymerase activity, and 3‘- --> 5' exonuclease activity). • The Klenow fragment of DNA polymerase I is widely used in molecular biology.

DNA-polymerase of eukaryotes :

Eukaryotes DNA polymerases have different nomenclature and activities are differentially distributed. These are : • DNA-pol α: initiate replication and synthesize primers. • DNA-pol β: replication with low fidelity. • DNA-pol γ: polymerization in mitochondria. • DNA-pol δ: elongation. • DNA-pol ε: proofreading and filling gap.

Primase :

• Also called DNAG. • Primers are short RNA fragments of a several nucleotides long. Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template. • Primers provide free 3´-OH groups to react with the α - P atom of dNTP to form phosphoester bonds. • Primase, DNAB, DNAC and an origin form a complex at the initiation phase.

• Helicase : Also referred to as DNAB, it opens the double strand DNA near the origin of replication with the assistance of DNAA and DNAC; and at the expense of ATP. • SSB protein : To avoid the recontact of the strands opened by helicase, single strand DNA binding protein attach to each single strand preventing the reformation of dsDNA. It also protects the vulnerable single stranded DNA against nucleases. • Topoisomerase II or DNA gyrase : Topoisomerase II are functional in releasing the torsional strain generated because of unwinding of the dsDNA. This also prevents the formation of supercoil ahead of replication forks. • DNA Ligase : Connects two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand. It also is responsible for sealing the nick in the process of replication, repairing, recombination, and splicing.