Understanding DNA Polymerases in Prokaryotic DNA Replication

in this video we are going to discuss about all the enzymes all the polymerases that are used for DNA

replication in prokaryotes now before we get to that point let's build up on the basics let's recap about the enzymes

which are involved in the replication process in bacteria so the key enzymes that are unwinding

the DNA are helices family enzymes now guy raised ensures that supercoiling is taken supercoiling problem is taken care

of now then Primus enzymes synthesize small RNA templates or RNA primers on which

the extension extension takes place with the help of these DNA polymerase enzymes now single strand binding protein

ensures that the strands which are opened up by the helices they don't intertwine which in each other it also

ensures that DNA polymerase 3 or DNA polymerase can perform its job and perform the polymerization part now DNA

paltry synthesize both the strands or extends both the strand from the end of the primer and the polymerization

Direction is 5 prime to 3 Prime in one strand the polymerization takes place in a uniform and uninterrupted fashion but

in the second strand the polymerization takes place in an interrupted and in and discontinuous fashion and the second

strand is known as lagging strand when well the continuous strand is known as the leading strand now let us look at

the type of DNA polymerase as we have discussed we have already introduced DNA polymerase 3 as a key replicating enzyme

I mean DNA polymerase 2 and 1 are other enzymes which are not the major ones but they are also very important DNA poultry

was not discovered before DNA pol one was the first one to be discovered and discovered by Arthur Kornberg but DNA

poultry was later recognized as the major enzyme teeny poultry has five prime to three prime polymerize activity

and 3 prime to 5 prime exonuclease activity that maybe it's proofreading DNA poll one has the similar activities

like the DNA poultry but it also has a five prime to three prime forward exonuclease activity and this is very

important for Nick translation and DNA repair DNA pol to has very similar activity just like DNA poultry but DNA

poultry is a major duplicating polymerase DNA pol - and DNA pol are actually used for repair and

sometimes DNA pol other DNA polymerase can also give a backup when DNA poultry is not there now let's take a look at

the active site of the DNA poultry to understand the process of this extension or the polymerization process now if you

zoom into the active site of the DNA pol 3 we would understand that new paces or the new nucleotides or dntps are added

and these addition takes place by a watson-crick base pairing rule right so here several round of addition of new

nucleotides are taking place and the extension is taking place in a 5 prime to 3 prime direction but in case of

lagging strand there are multiple rounds by which the DNA polymerase has to synthesize the strand and that's why

this synthesis of the Strand is discontinuous and interrupted now we should ask that

what is the mechanism of catalysis of this kind of DNA polymerase enzymes and the key answer is it's a metal ion

catalysis so let's look at it in the active site of the DNA polymerase 3 you can see there are several exported

residues with which magnesium ions are linked by ionic interaction now this magnesium ions are very important

because magnesium ions actually neutralize the charge on the negative phosphate backbone now making it more

viable for a nucleophilic attack from this hydroxyl group of the new incoming incoming nucleotide and that is

how the polymerization is taking place and the phosphodiester bond is actually created and also the nucleotide is

interacting with the complementary strand nucleotide and in between them the hydrogen bond is forming and all is

taking place in the active site now whenever there is a proper match these nucleotides are nicely placed in the

active site of the enzyme and this reaction can take place but in case of mismatch let's say a faulty nucleotide

or a nucleotide which should not pair with this particular nucleotide is there in that situation the 3d positioning of

these nucleotide or this dntp is not proper into the group of this DNA polymerase and that's create a steric

repulsion and in a three-dimensional structure they are quite far away from each other to start a nucleophilic

attack and that is why in case of mismatch the polymerization halts until it is repaired so the exonuclease axial

activity then kicks in and try to repair this kind of mismatch also we should not try to understand why only dntps

are used by DNA polymerase not the NT piece or the entities that are important for RNA synthesis right now

deoxyribonucleotide can nicely fit into the active site whereas RN tepees can not fit nicely into the active site of

this enzyme as a result it is always an inner signal when our ntp is entering the nucleotide binding groove so DNA

polymerase can distinguish between dntps and the r NT piece even though our ntp is at 10 times higher in concentration

compared to dntps only dnt bees are preferred by DNA polymerase not darn tepees now whenever there is a mismatch

the wrong nucleotide creates a bump in the DNA and that is recognized by the polymerase and polymerase will move in a

backward direction to get rid of it remember the active site in which the polymerization reaction is taking place

and the active site where the exonuclease activity is is a complete different site and the exonuclease site

try to get the base out of it and allow new new incoming nucleotide to be paired in a proper location and that is how the

exonuclease activity works but about exonuclease activity I have a total different different different video so I

would suggest you to watch that but for that it was just an overview of all type of DNA polymerase that are presenting

the prokaryotes and how they roughly work and how they are different from each other so I hope you enjoyed this

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