🦠Cell Biology Unit 13 – DNA Replication and Repair

Key Concepts in DNA Structure

• DNA consists of two antiparallel polynucleotide strands wound around each other to form a double helix
• Each strand is composed of a sugar-phosphate backbone with nitrogenous bases attached to the sugar molecules
• The four nitrogenous bases in DNA include adenine (A), thymine (T), guanine (G), and cytosine (C)
  • Adenine pairs with thymine through two hydrogen bonds
  • Guanine pairs with cytosine through three hydrogen bonds
• The complementary base pairing (A-T and G-C) allows for the precise replication and transmission of genetic information
• The sugar in DNA is deoxyribose, which differs from the sugar in RNA (ribose) by the absence of an oxygen atom at the 2' position
• The phosphate groups in the backbone provide structural stability and a negative charge to the DNA molecule
• The double helix structure is maintained by hydrogen bonds between base pairs and base stacking interactions between adjacent bases

The DNA Replication Process

• DNA replication is the process by which a cell duplicates its genetic material before cell division
• Replication begins at specific sites called origins of replication, where the double helix is unwound by helicases
• The unwinding of the double helix creates a replication fork, which consists of two single-stranded DNA templates
• DNA primase synthesizes short RNA primers complementary to the single-stranded DNA templates, providing a starting point for DNA synthesis
• DNA polymerases extend the primers by adding nucleotides complementary to the template strands in the 5' to 3' direction
  • DNA polymerase III is the main enzyme responsible for DNA synthesis in prokaryotes
  • In eukaryotes, DNA polymerase α, δ, and ε are involved in replication
• The leading strand is synthesized continuously in the 5' to 3' direction, while the lagging strand is synthesized discontinuously as Okazaki fragments
• Okazaki fragments are later joined together by DNA ligase to form a continuous strand
• As replication proceeds, the replication forks move bidirectionally from the origin until they meet and terminate

Enzymes and Proteins Involved

• Helicases unwind the double helix by breaking the hydrogen bonds between base pairs, creating single-stranded DNA templates for replication
• Topoisomerases (DNA gyrase in prokaryotes and topoisomerase II in eukaryotes) relieve the tension and supercoiling caused by the unwinding of the double helix
• Single-stranded DNA binding proteins (SSBs) stabilize the single-stranded DNA templates and prevent them from reannealing
• DNA primase synthesizes short RNA primers (8-12 nucleotides) complementary to the single-stranded DNA templates
• DNA polymerases (III in prokaryotes, α, δ, and ε in eukaryotes) extend the primers by adding nucleotides complementary to the template strands
  • DNA polymerases have proofreading activity (3' to 5' exonuclease) to ensure high fidelity of replication
• DNA ligase seals the nicks between Okazaki fragments on the lagging strand, creating a continuous strand
• Sliding clamp proteins (β-clamp in prokaryotes and PCNA in eukaryotes) encircle the DNA and tether the polymerases to the template, increasing processivity

Replication Origins and Directionality

• DNA replication begins at specific sites called origins of replication, which are recognized by initiator proteins
• In prokaryotes, there is typically a single origin of replication (oriC) per circular chromosome
  • The oriC contains multiple copies of a consensus sequence that is recognized by the DnaA initiator protein
• In eukaryotes, there are multiple origins of replication distributed throughout the linear chromosomes
  • The origins are called autonomously replicating sequences (ARS) in yeast and origin recognition complex (ORC) binding sites in higher eukaryotes
• Replication proceeds bidirectionally from each origin, with two replication forks moving in opposite directions
• The leading strand is synthesized continuously in the 5' to 3' direction, while the lagging strand is synthesized discontinuously as Okazaki fragments
  • Okazaki fragments are 1,000-2,000 nucleotides long in prokaryotes and 100-200 nucleotides long in eukaryotes
• Termination of replication occurs when two replication forks converge and the remaining single-stranded gaps are filled and ligated

DNA Repair Mechanisms

• DNA damage can occur due to various factors, including UV radiation, chemical mutagens, and reactive oxygen species
• Cells have evolved several DNA repair mechanisms to maintain genomic integrity and prevent mutations
• Base excision repair (BER) corrects small, non-helix-distorting lesions such as oxidized or deaminated bases
  • DNA glycosylases recognize and remove the damaged base, creating an apurinic/apyrimidinic (AP) site
  • AP endonuclease cleaves the phosphodiester backbone at the AP site, and DNA polymerase β fills the gap
• Nucleotide excision repair (NER) corrects bulky, helix-distorting lesions such as UV-induced pyrimidine dimers and chemical adducts
  • The damage is recognized by the XPC-HR23B complex, and the TFIIH complex unwinds the DNA around the lesion
  • Endonucleases (XPG and ERCC1-XPF) excise a 24-32 nucleotide segment containing the damage, and DNA polymerase δ or ε fills the gap
• Mismatch repair (MMR) corrects base-base mismatches and insertion/deletion loops that escape the proofreading activity of DNA polymerases
  • The MutS protein recognizes the mismatch, and MutL recruits the MutH endonuclease to nick the newly synthesized strand
  • Exonucleases degrade the nicked strand, and DNA polymerase III resynthesizes the correct sequence
• Double-strand break repair mechanisms include homologous recombination (HR) and non-homologous end joining (NHEJ)
  • HR uses the sister chromatid as a template to repair the break accurately, while NHEJ directly ligates the broken ends, which can introduce mutations

Mutations and Their Consequences

• Mutations are permanent changes in the DNA sequence that can arise from errors during replication or unrepaired DNA damage
• Point mutations involve the substitution, insertion, or deletion of a single nucleotide
  • Substitutions can be transitions (purine to purine or pyrimidine to pyrimidine) or transversions (purine to pyrimidine or vice versa)
  • Insertions and deletions can cause frameshift mutations if they are not a multiple of three nucleotides
• Mutations in coding regions can be silent (no change in amino acid), missense (change in amino acid), or nonsense (premature stop codon)
• Mutations in regulatory regions can affect gene expression by altering transcription factor binding sites or promoter sequences
• Chromosomal mutations involve large-scale changes in chromosome structure, such as deletions, duplications, inversions, and translocations
• Mutations can be spontaneous (occurring without any apparent cause) or induced by mutagens (physical or chemical agents that increase the mutation rate)
• Accumulation of mutations can lead to genetic diseases, cancer, and evolutionary changes in populations

Regulation of DNA Replication

• DNA replication is tightly regulated to ensure that the genome is duplicated accurately and only once per cell cycle
• In prokaryotes, the initiation of replication is controlled by the DnaA protein, which binds to the oriC and recruits the replication machinery
  • The activity of DnaA is regulated by its ATP/ADP-bound state and the availability of hemimethylated GATC sites in the oriC
• In eukaryotes, the initiation of replication is controlled by the assembly of the pre-replication complex (pre-RC) at the origins
  • The ORC binds to the origins and recruits Cdc6 and Cdt1, which load the MCM helicase complex onto the DNA
  • The MCM complex is activated by the Dbf4-dependent kinase (DDK) and cyclin-dependent kinases (CDKs) to initiate replication
• The cell cycle checkpoints (G1/S, intra-S, and G2/M) ensure that replication is completed before cell division and prevent re-replication
  • The checkpoints are mediated by CDKs and the ATR/ATM kinases, which sense DNA damage and stalled replication forks
• Epigenetic modifications, such as DNA methylation and histone modifications, can also regulate the timing and efficiency of replication
  • Heavily methylated regions (heterochromatin) tend to replicate later in S phase compared to euchromatic regions

Clinical and Research Applications

• Understanding the mechanisms of DNA replication and repair has important implications for human health and disease
• Defects in DNA repair genes can lead to hereditary cancer syndromes, such as Lynch syndrome (MMR defects) and xeroderma pigmentosum (NER defects)
  • Identifying mutations in these genes can help in early diagnosis, prevention, and targeted therapies
• Inhibitors of DNA replication enzymes (e.g., DNA polymerases, topoisomerases) are used as anticancer and antiviral drugs
  • Examples include cisplatin (forms DNA adducts), 5-fluorouracil (inhibits thymidylate synthase), and etoposide (inhibits topoisomerase II)
• DNA replication and repair enzymes are also targets for the development of new antimicrobial agents, as they are essential for bacterial survival
• Techniques such as DNA fiber autoradiography and fluorescence microscopy have enabled the visualization and study of replication dynamics in living cells
• Next-generation sequencing technologies have revolutionized the field of genomics, allowing for the rapid and cost-effective sequencing of entire genomes
  • These technologies rely on the principles of DNA replication, such as the use of DNA polymerases and primers
• Genome editing tools, such as CRISPR-Cas9, have emerged as powerful methods for studying gene function and developing gene therapies
  • These tools exploit the cellular DNA repair mechanisms (NHEJ and HR) to introduce targeted modifications in the genome