DNA - An overview

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Slide 68

When an unpaired or incorrectly paired base are clip off by exonucleases.

When an appropriate base-paired terminus results, polymerase begins resynthesis by adding nucleotides to the 3’ end.

The 5’ to 3’ exonuclease activity of many prokaryotic DNA polymerases is also very important.

It functions in the removal of segments of DNA damaged by UV and other agents.

Slide 69

Analogous to RNA, DNA is synthesized from deoxynucleoside 5-triphosphate precursors (dNTPs).

The enzyme requires the 5’triphosphates of each of the four deoxyribonucleosides:

dATP : deoxyadenosine triphosphate

dTTP: deoxythymidine triphosphate (TTP)

dGTP: deoxyguanosine triphosphate

dCTP: deoxycytidine triphosphate

Slide 70

This enzyme is active only in the presence of Mg+ ions and preexisting DNA.

This DNA must provide two essential components, one serving a primer function and other a template function.

1. Primer DNA: DNA polymerase I cannot initiate the synthesis of de novo. It has an absolute requirement for a free 3’hydroxyl on preexisting DNA chain.

DNA Polymerase I catalyzes the formation of a phosphodiester bridge between the 3’OH at the end of the primer DNA chain and 5’phosphate of the incoming deoxyribonucelotide.

The direction of synthesis is always 5’ to 3’

2. Template provides ssDNA that will direct the addition of each complementary deoxynuceotide

Slide 71

“Replicating Apparatus” is complex

DNA replication is complex.

It is carried out by multienzyme complex, often called, replication apparatus or the replisome.

In eukaryotes, the components of replication machinery are just beginning to be identified.

Even in prokaryotes, DNA replication requires many different proteins

Slide 72

Replication fork: The junction between the newly separated strands and unreplicated double stranded DNA

Leading and Lagging strand: Due to the anti-parallel nature of DNA, one strand will synthesis continuously towards replication fork and other strand will synthesis discontinuously away from the replication fork.

The continuously synthesizing strand is called leading strand and discontinuously synthesizing strand is called lagging strand.

Okazaki fragment: A short fragment of DNA formed on the lagging strand during replication is called Okazagi fragment. It will be around 100 – 1000 bp in length. In eukaryotes it identified about 100-200 nucleotides length.

Processivity: The ability of an enzyme to catalyze many reactions before releasing its substrate is called processsivity