Cell division involves the duplication of the entire DNA so that two genetically identical daughter cells arise from a single cell. DNA is bound to proteins in the nucleus and is tightly packed. The duplication of DNA (DNA replication) requires that the DNA is loosened and the double helix is unwound. Specific proteins, including DNA polymerase, then synthesize a complementary daughter strand at each single strand. Two double DNA strands are formed, each with one new and one original strand. The process of DNA replication includes control mechanisms to keep the genetic information as stable as possible, but errors, such as the incorporation of the wrong base, still occur. External factors as well as internal cellular processes lead to alterations in the chemical structure of DNA. If DNA errors are not repaired, mutations and/or cell destruction may occur. DNA repair mechanisms are thus important to ensure a sufficient degree of genomic stability.
Fundamentals of DNA replication
- Purpose: To ensure that daughter cells contain genetically identical information to the parent cells by copying double-stranded DNA (dsDNA) during cell division (of the S phase of the cell cycle)
- Is semiconservative: Replication results in two identical dsDNA molecules, each consisting of a parent strand (serves as a template) and a newly synthesized daughter strand.
- Occurs in the 5' → 3' direction
- Is bidirectional: Replication occurs in both directions of the original dsDNA (i.e., the direction of each parent strand).
- Occurs in three stages :
- Involves many proteins/enzymes
|Helicase||Unwind local segments of the DNA double helix (ATP-dependent reaction) at the replication fork||DnaB||MCM complex|
|Single-stranded DNA-binding proteins (SSBs)||Prevents reannealing of separated strands||SSB (single-strand binding protein)||RPA (replication protein A)|
|Type I topoisomerase|| |
Cleaves only one of both DNA strands
|Topoisomerase I (type IA topoisomerases)||Topoisomerase I (type IB topoisomerases)|
|Type II topoisomerase|| || |
Topoisomerase II (DNA gyrase) and topoisomerase IV
|Primase (DNA-dependent RNA polymerase)||Synthesis of short RNA sequences (RNA primers)||Primase (DnaG)||Primase activity as a component of DNA polymerase α|
|DNA-dependent DNA polymerase||Extends RNA primers by adding 50–100 nucleotides||∅||Polymerase α|
|Replicates the lagging strand||DNA polymerase III||Polymerase δ|
|Extend the leading strand||Polymerase ε|
|Removal of primers||RNase H and DNA polymerase I (5'→3' exonuclease activity)||RNase H and FEN-1 (flap endonuclease-1)|
|Gaps between fragments are filled after primer removal||DNA polymerase I||Polymerase δ|
|Ligase||Links newly synthesized DNA fragments (ATP or NAD+-dependent reaction) by catalyzing the formation of phosphodiester bonds||DNA ligase||DNA ligase|
|Telomerase||Ensures complete replication of the ends of linear chromosomes||∅||Telomerase|
The process of DNA replication
- Specific proteins (comprising a prepriming complex) recognize and bind to the origin of replication
- At the ori, helicase separates and begins unwinding dsDNA into single strands, forming 2 replication forks
- SSBs prevent the single strands from reannealing and protects ssDNA from cleavage
- Supercoil relaxation: DNA topoisomerases relieve overwinding (positive supercoils) or underwinding (negative supercoils) that develop during DNA separation and elongation.
- Primer synthesis: : Primase synthesizes a short, 5–10 nucleotide long RNA primer that is complementary to the template strand
DNA synthesis: For simultaneous replication of both parent strands, DNA replication occurs continuously on the leading strand and discontinuously on the lagging strand in a 5'→3' direction as complementary deoxynucleotides are added to the free 3'OH group of the daughter strand
Reaction catalyzed by DNA polymerase (polymerase δ in eukaryotes, DNA polymerase III in prokaryotes)
- 5'→3' DNA polymerase activity catalyzes the nucleophilic attack of the 3'OH group to the α-phosphate of the incoming deoxynucleotide triphosphate, forming an ester bond
- Single strands are elongated by a single nucleotide.
- Pyrophosphate (PPi) is released
- Pyrophosphate is cleaved into two phosphates by pyrophosphatase.
Leading strand: A complementary daughter strand of DNA whose replication is continuous due to its free 3'OH end (initially on the primer)
- Only one primer is required.
- Lagging strand: A complementary daughter strand of DNA whose replication is discontinuous as new RNA primers are constantly being synthesized at the moving replication fork to ensure a free 3'OH end for elongation.
- Reaction catalyzed by DNA polymerase (polymerase δ in eukaryotes, DNA polymerase III in prokaryotes)
- Proofreading: Some polymerases (e.g., DNA polymerase I and III) have 3'→5' exonuclease activity and remove incorrectly paired nucleotides.
- Primer removal: RNA primers are excised in the opposite direction of synthesis (so 5'→3')
- Filling the gaps: During primer removal, DNA polymerase fills the gaps with deoxynucleotides complementary to the parent strand until the free ends meet.
- Joining of the ends
- Structure: a non-coding DNA fragment of several thousand bp (composed of tandem repeats of TTAGGG) at the 3' ends of chromosomes
Prevent the loss of structural genes during replication of linear DNA double-strands
- The lagging strand becomes shorter with each process of DNA replication.
- Telomeres extend the cell's lifespan by slowing the cell's aging process
- Prevent the loss of structural genes during replication of linear DNA double-strands
- Maintenance: by telomerase
Endogenous sources of DNA errors
- Replication errors: Repetitive sequences and
- Depurination: thermal cleavage of the N-glycosidic bond between deoxyribose and a purine base at body temperature
- Free radical damage: highly reactive oxygen or hydroxyl radicals that cause oxidative stress and can chemically alter bases
- Spontaneous oxidative deamination
Exogenous sources of DNA errors
- Alkylating substances: methylate or ethylate bases, causing them to pair with other bases than the usual
- Intercalating substances: embed between the stacked DNA base pairs, causing replication to stop and increasing the risk of strand breaks
- Examples: ethidium bromide, acridine dye, dactinomycin
- UV radiation (both UVA and UVB) can result in dimer formation of neighboring pyrimidine bases (pyrimidine dimers)
- Can lead to ssDNA and dsDNA breaks
- Also results in the increased formation of free radicals
|Type of DNA repair||Mechanisms||Notable conditions if defective|
|ssDNA repair||Base excision repair|| || |
|Nucleotide excision repair|
|DNA mismatch repair|
|Nonhomologous end joining|| |
|Homologous end joining|