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LET'S TALK LIFE SCIENCE MOLECULAR BIOLOGY

Suraj Prakash Sharma | Ekta Chotia

DNA REPLICATION
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1.9.1.     Rolling-circle replication

It involves a single stranded intermediate that seems to roll off the plasmid. Plasmid’s double stranded DNA has positive and negative strand. In rolling circle replication double stranded ori that place within stem-loop structure get nicked by Rep A protein at positive strand and free 3’ OH serve as a primer which get replicate by DNA pol III  in leading strand manner and displacing the 5’ end which is non-template strand by strand displacement reaction. The ends of the displaced strand are ligated to make a single stranded circular DNA molecule. RNA polymerase makes a primer at the 5’ end containing strand, thus also capable to act as template for lagging strand synthesis. After synthesis of new strand primer get remove by DNA pol I. The ends of the displaced strand are ligated by ligase to make a second double- stranded plasmid. E.g. M13, Φ 174, F plasmid.

F-Plasmid replication

Most plasmids use one of two common mechanisms of replication, both of which depend on the host cell replicative machinery for replication (DNA polymerases, ligases, primases, helicases, etc. are usually supplied by the host cell).

1.9.2.     Theta replication –

It is normal mode of DNA replication which resembles the greek letter theta (θ) during replication. The theta mode of DNA replication is bidirectional in nature.

1.9.3.     Linear replication –

This the type of replication takes place in linear chromosome like in eukaryotes.

1.9.4.     Replication of organelle DNA (Models for mtDNA replication)

DNA polymerase γ is used exclusively for mtDNA replication and proofreading. Two models have been proposed for the mode of mtDNA replication, 1) strand displacement model, appeal to continuous DNA replication and 2) coupled model, proposes semidiscontinous DNA replication.

Strand displacement model

The strand displacement model (also called the strand asynchronous model) for mammalian mtDNA replication is the most widely accepted, longest standing model. In strand displacement model, replication is unidirectional around the circle and there is one replication fork for each strand in which One  strand is called the heavy strand (H) and other strand is called light strand (L). The designation of the strands as H and L due to their different buoyant densities in denaturing CsCl density gradients centrifugation. Two origins (OH and OL) because here one priming event per template strand and it start within a region called the displacement or “D” loop of 500–600 nucleotide. The RNA primer synthesis by mitochondrial RNA polymerase. Firstly replication start on H strand starting at OH. When DNA polymerase polymerise approximately two-thirds of the mtDNA as a result replication fork get passes the major origin of L strand synthesis,  thus exposing the site in single-stranded form at which new H strand synthesis start from OL. Synthesis is continuous around the circle on both strands. Multifunction endoribonuclease, RNase MRP cleave RNA primer. Same mechanism for cpDNA (cytoplasmic DNA) replication.

Strand coupled model

In strand coupled model at multiple sites lagging strand replication (L strand) get initiated, showing discontinuous synthesis of short Okazaki fragments thus requiring multiple primers. So, in this model the coupled lagging strand and leading (H strand) synthesis represents a semidiscontinous, bidirectional mode of DNA replication. The endoribonuclease, RNase MRP (mitochondrial RNA processing) has at least two functions, 1) removal of RNA primers in mtDNA replication, 2) pre-ribosomal RNA’s nucleolar processing. Mutation in RNase MRP cause cartilage-hair hypoplasia a rare form of dwarfism and due to its consequences  multiple phenotypic manifestations (pleiotropy) occur which included short limbs, short stature, fine sparse hair, impaired cellular immunity, anemia, and predisposition to several cancers.

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