Replication Basics

To gain a solid understanding of Molecular Diagnostics, one has to grasp the fundamentals of DNA Replication. The double helix nature of DNA requires numerous moving parts working together to produce two identical strands of DNA from one original DNA molecule.

Image courtesy of http://genmed.yolasite.com/fundamentals-of-genetics.php
Image courtesy of http://genmed.yolasite.com/fundamentals-of-genetics.php

The Origin of Replication

The semi-conservative process of DNA replication occurs in a 5’ to 3’ anti-parallel direction. The replication process is described as semi-conservative because the sequence of nucleotides is maintained through new generations of replication. An extremely important enzyme involved in the beginning stages of DNA replication, is called Topoisomerase. It is responsible for regulating the over-winding and under-winding of DNA just ahead of the replication fork. Topoisomerase binds to the DNA then “cuts” the phosphate backbone so that the DNA can be unwound then resealed at the end of replication. Also, before replication can begin, an enzyme called helicase must first unwind and untangle the double-stranded DNA. Single stranded binding proteins (ssbp) prevent premature binding as well as protect the single stranded DNA from being digested by nucleases.

Leading Strand vs. Lagging Strand

During replication, two separate strands of DNA are formed in different ways. The lagging strand exhibits discontinuous 3’ to 5’ growth away from the replication fork and requires primase to “prime” the synthesis of the lagging strand. An RNA primer is added to the lagging strand of the DNA by RNA polymerase. This RNA primer begins synthesis of the lagging strand. The separate fragments of the lagging strand are termed Okazaki fragments. It’s important to note that due to the discontinuous formation of the lagging strand, each Okazaki fragment requires its own, separate, RNA primer. Finally, DNA ligase forms phophodiester bonds between the existing DNA strands to join the Okazaki fragments together. Alternatively, the leading strand during replication grows towards the replication fork in a 5’ to 3’ direction. The leading strand only needs one single RNA primer to immediately begin replication and therefore does not require DNA ligase.

LEADING STRAND SYNTHESIS REQUIREMENTS
Toward Replication Fork Single RNA Primer
5’→ 3’
Continuous Growth
LAGGING STRAND SYNTHESIS REQUIREMENTS
Away from Replication Fork Primase
3’→ 5’ Multiple RNA Primers
Discontinuous Growth DNA Ligase
Creation of Okazaki Fragments

DNA Polymerase III and its Role in Replication

While you should become familiar with the extensive list of DNA Polymerases (shown below), the core polymerase involved in DNA replication is DNA Polymerase III. It functions as a catalyst in the formation of the phosphodiester bonds between an incoming deoxyribose nucleotide triphosphate (dNTP) determined by hydrogen bonding to the template at the 3’ end of the primer.

PROKARYOTIC DNA POLYMERASES FUNCTION
DNA Polymerase I Recombination, Repair, Replication
DNA Polymerase II Repair
DNA Polymerase III Core Polymerase

Replication

DNA Polymerase IV and V Bypass DNA Damage (Y-Family DNA Polymerases)
EUKARYOTIC DNA POLYMERASES FUNCTION
Alpha (α) RNA Primase

Lagging Strand

Replication (Initiation, Okazaki Fragment Priming)

Beta (β) DNA Repair
Delta (δ) Leading Strand

Repair

Epsilon (ε) Sensor of DNA replication that coordinates transcription cycle

Repair

Gamma (γ) Mitochondrial Replication
RNA POLYMERASES FUNCTION
RNA Polymerase I rRNA (ribosomal RNA)
RNA Polymerase II mRNA (messenger RNA)
RNA Polymerase III tRNA (transfer RNA)

sbRNA (small nuclear RNA)

L Noll Image_small

-LeAnne Noll, BS, MB(ASCP)CM is a molecular technologist at Children’s Hospital of Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.

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