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.
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|
|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
|DNA Polymerase IV and V||Bypass DNA Damage (Y-Family DNA Polymerases)|
|EUKARYOTIC DNA POLYMERASES||FUNCTION|
|Alpha (α)||RNA Primase
Replication (Initiation, Okazaki Fragment Priming)
|Beta (β)||DNA Repair|
|Delta (δ)||Leading Strand
|Epsilon (ε)||Sensor of DNA replication that coordinates transcription cycle
|Gamma (γ)||Mitochondrial Replication|
|RNA Polymerase I||rRNA (ribosomal RNA)|
|RNA Polymerase II||mRNA (messenger RNA)|
|RNA Polymerase III||tRNA (transfer RNA)
sbRNA (small nuclear RNA)
-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.