What is the process of DNA replication?

DNA Replication: A Comprehensive Overview

DNA replication is a highly conserved, tightly controlled process that occurs during S-phase of the cell cycle, performed by DNA polymerase enzymes to duplicate the entire genome before cell division 1.

The Fundamental Process

DNA replication is inherently error-prone, requiring multiple quality control mechanisms to maintain genomic integrity. 1

Core Mechanism

• DNA polymerases replicate DNA during S-phase but are not mistake-free 1
• Single-nucleotide variations (SNVs) result from base incorporation errors, while polymerase slippages cause insertions and deletions (indels), particularly in repetitive genomic segments called microsatellites 1
• The replication machinery involves coordinated action of at least two different DNA polymerases, single-stranded DNA-binding proteins, clamp-loading complexes, and polymerase clamps 2

Initiation of Replication

• Replication initiation involves initiator proteins interacting with origins of replication in DNA, followed by regulated assembly of two replisome complexes at each origin 3
• In eukaryotes, replication must initiate from multiple origins across the genome, requiring coordination to ensure the genome replicates exactly once per cell division cycle 4
• Unlike bacteria with readily identifiable origin sequences, eukaryotic replication origins are more complex and context-dependent 5

Quality Control Mechanisms

Replication fidelity is governed by two primary systems: the exonuclease proofreading domains of DNA polymerases (encoded by POLD1 and POLE genes) and the mismatch repair (MMR) system 1

Proofreading Systems

• The exonuclease domains in DNA polymerases provide the first line of defense against replication errors 1
• The MMR system, consisting of proteins encoded by PMS2, MSH6, MLH1, and MSH2 genes, provides secondary error correction 1
• These quality control mechanisms are essential to prevent mutation accumulation in dividing cells 1

DNA Repair and Adduct Recognition

• DNA repair systems preferentially eliminate lesions localized in the coding (transcribed) strand over the non-transcribed strand 1
• Adduct recognition and removal rates depend on the bases surrounding the damaged base 1
• The majority of mutations in cells occur when DNA adducts are not removed before replication 1

Coordination with Cell Cycle

DNA replication must be tightly coupled to cell-cycle progression and DNA repair mechanisms to maintain genome integrity 2

Regulatory Integration

• Replication is coordinated with inheritance of chromatin structure, developmental patterning in multicellular organisms, and cell division 4
• Once-per-cell-cycle regulation ensures each genomic region replicates exactly once before mitosis 5
• The process involves DNA polymerase-switching mechanisms during Okazaki fragment synthesis on the lagging strand 2

Leading and Lagging Strand Synthesis

Replication occurs bidirectionally from origins, with continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand 2

Okazaki Fragment Processing

• Lagging strand synthesis produces Okazaki fragments that require specific nucleases, helicases, and DNA ligase for maturation 2
• This process involves recruitment of specialized enzymes to join the fragments into continuous DNA 2

Clinical Significance of Replication Errors

When replication fidelity mechanisms fail due to germline mutations in MMR or polymerase proofreading genes, uncontrolled mutagenesis and genomic instability result, leading to cancer predisposition syndromes 1

Replication Repair Deficiency

• Aberrations in MMR and polymerase-proofreading genes cause high tumor mutation burden (TMB) and microsatellite instability (MSI), the hallmarks of replication repair deficiency (RRD) 1
• Biallelic mutations in MMR genes (MSH2, MSH6, MLH1, PMS2) cause constitutional mismatch repair deficiency (CMMRD), one of the most aggressive childhood cancer predisposition syndromes 1
• Point mutations and microsatellite instability are the defining features of replication repair deficiency 1

Technical Considerations

DNA replication faces technical variability during sequencing and analysis that must be accounted for in genomic studies 1

Experimental Variation

• Technical variability arises from sequencing platforms, flow cell differences, and random sampling variance of the sequencing process 1
• Variations in library preparation introduce additional experimental noise even when protocols remain identical 1
• Bioinformatics tools must accommodate experimental variation to generate consistent results across different sequencing runs 1

Conservation Across Life

The core DNA replication machinery is conserved across all three domains of life (bacteria, archaea, and eukaryotes), though with varying complexity 3, 4

• Archaea and eukaryotes share homologous replication initiation systems, while bacteria use analogous but non-homologous mechanisms 3
• Despite differences in origin specification, the regulatory proteins controlling replication are highly conserved from yeast to humans 5