🔬Biological Chemistry I Unit 12 – DNA Replication and Repair

DNA Structure Basics

• DNA consists of two antiparallel polynucleotide strands that form a double helix structure
  • Each strand is composed of a sugar-phosphate backbone with nitrogenous bases attached
  • The sugar in DNA is deoxyribose, which lacks a hydroxyl group at the 2' position compared to ribose in RNA
• The four nitrogenous bases in DNA are adenine (A), thymine (T), guanine (G), and cytosine (C)
  • Purines (A and G) have a double-ring structure, while pyrimidines (T and C) have a single-ring structure
• Complementary base pairing occurs between the two strands through hydrogen bonding
  • A pairs with T via two hydrogen bonds, while G pairs with C via three hydrogen bonds
  • The specific base pairing maintains the uniform width of the DNA double helix
• The double helix structure is further stabilized by base stacking interactions between adjacent bases
• The directionality of each DNA strand is determined by the orientation of the sugar-phosphate backbone
  • The 5' end has a free phosphate group, while the 3' end has a free hydroxyl group
• The antiparallel nature of the two strands means that they run in opposite directions (5' to 3' and 3' to 5')

Replication Process Overview

• DNA replication is the process by which a cell duplicates its genetic material before cell division
• Replication ensures that each daughter cell receives an identical copy of the genetic information
• The process is semiconservative, meaning that each newly synthesized DNA molecule contains one original strand and one new strand
• Replication begins at specific sites along the DNA called origins of replication
  • In prokaryotes, there is typically a single origin of replication, while eukaryotes have multiple origins
• The DNA double helix unwinds and separates into two single strands, each serving as a template for the synthesis of a new complementary strand
• 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 short Okazaki fragments
• DNA polymerases catalyze the addition of nucleotides to the growing DNA strands, using the parental strands as templates
• After replication, each daughter DNA molecule consists of one original strand and one newly synthesized strand

Key Enzymes in DNA Replication

• DNA helicase unwinds the double helix by breaking the hydrogen bonds between complementary base pairs
  • It moves along the DNA in the 5' to 3' direction, separating the two strands to create a replication fork
• Single-stranded DNA binding proteins (SSBs) bind to the exposed single-stranded DNA to prevent the strands from reannealing and to protect them from degradation
• DNA primase synthesizes short RNA primers (8-12 nucleotides) that provide a starting point for DNA synthesis
  • Primers are necessary because DNA polymerases cannot initiate synthesis de novo
• DNA polymerases catalyze the addition of nucleotides to the growing DNA strands
  • DNA polymerase III is the main replicative polymerase in prokaryotes, while DNA polymerases α, δ, and ε are involved in eukaryotic replication
  • These polymerases have a high processivity and fidelity, ensuring accurate and efficient DNA synthesis
• DNA ligase seals the nicks between Okazaki fragments on the lagging strand, creating a continuous strand of DNA

Replication Machinery and Mechanisms

• The replisome is a multi-protein complex that carries out DNA replication at the replication fork
  • It consists of DNA helicase, primase, DNA polymerases, and other accessory proteins
• The leading strand is synthesized continuously by DNA polymerase in the 5' to 3' direction, following the movement of the replication fork
• The lagging strand is synthesized discontinuously as Okazaki fragments due to the antiparallel nature of DNA and the 5' to 3' synthesis by DNA polymerases
  • Primase synthesizes RNA primers at regular intervals along the lagging strand
  • DNA polymerase extends the primers, synthesizing the Okazaki fragments in the 5' to 3' direction
  • The RNA primers are later removed and replaced with DNA by DNA polymerase I in prokaryotes or DNA polymerase δ in eukaryotes
• Sliding clamps (β clamp in prokaryotes, PCNA in eukaryotes) encircle the DNA and tether the DNA polymerases to the template, increasing processivity
• Topoisomerases (DNA gyrase in prokaryotes, topoisomerases I and II in eukaryotes) relieve the positive supercoiling that builds up ahead of the replication fork
  • They introduce temporary single-strand or double-strand breaks in the DNA to release the torsional stress

DNA Damage Types

• Deamination occurs when the amino group is removed from a nitrogenous base, converting cytosine to uracil or adenine to hypoxanthine
• Depurination and depyrimidination involve the hydrolysis of the glycosidic bond between the sugar and the purine or pyrimidine base, resulting in an apurinic or apyrimidinic (AP) site
• Oxidative damage is caused by reactive oxygen species (ROS) that can modify bases, such as the formation of 8-oxoguanine from guanine
• Alkylation involves the addition of alkyl groups to bases, which can interfere with base pairing and lead to mutations
• Pyrimidine dimers, such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, are formed when adjacent pyrimidines become covalently linked due to UV radiation exposure
• Double-strand breaks (DSBs) occur when both strands of the DNA are severed, often caused by ionizing radiation or certain chemicals
• Interstrand crosslinks are covalent bonds formed between the two strands of the DNA, preventing their separation during replication and transcription

DNA Repair Mechanisms

• Base excision repair (BER) corrects small, non-helix-distorting lesions such as oxidized or deaminated bases and AP sites
  • Glycosylases recognize and remove the damaged base, creating an AP site
  • AP endonuclease cleaves the phosphodiester backbone at the AP site
  • DNA polymerase fills in the missing nucleotide, and DNA ligase seals the nick
• Nucleotide excision repair (NER) repairs bulky, helix-distorting lesions such as pyrimidine dimers and intrastrand crosslinks
  • The damage is recognized, and a segment of the strand containing the lesion is excised
  • DNA polymerase fills in the gap using the complementary strand as a template, and DNA ligase seals the nick
• Mismatch repair (MMR) corrects mismatched base pairs and small insertion/deletion loops that arise during replication
  • The mismatch is recognized, and a segment of the newly synthesized strand containing the error is excised
  • DNA polymerase fills in the gap using the parental strand as a template, and DNA ligase seals the nick
• Double-strand break repair occurs through two main pathways: non-homologous end joining (NHEJ) and homologous recombination (HR)
  • NHEJ directly ligates the broken ends, which can result in small insertions or deletions
  • HR uses the sister chromatid as a template to repair the break accurately, but it is limited to the S and G2 phases of the cell cycle

Replication Errors and Mutations

• Replication errors can occur due to the incorporation of incorrect nucleotides by DNA polymerases
  • DNA polymerases have proofreading activity that allows them to remove misincorporated nucleotides, reducing the error rate
• Slippage of the DNA polymerase can lead to the formation of small insertions or deletions, particularly in repetitive sequences (microsatellites)
• Unrepaired DNA damage can lead to mutations during replication, as the polymerase may incorporate an incorrect nucleotide opposite the damaged base
• Translesion synthesis (TLS) polymerases can bypass certain types of DNA damage, but they have a higher error rate than replicative polymerases
• Mutations can be classified as substitutions (point mutations), insertions, or deletions
  • 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 occur in coding regions and the number of bases added or removed is not a multiple of three
• Mutations can have various effects on gene function, such as silent mutations (no change in amino acid), missense mutations (change in amino acid), nonsense mutations (premature stop codon), or frameshift mutations (altered reading frame)

Clinical and Research Applications

• Mutations in genes involved in DNA replication and repair can lead to genetic instability and an increased risk of certain cancers (hereditary nonpolyposis colorectal cancer, HNPCC)
• Inhibitors of DNA replication enzymes, such as DNA polymerases and topoisomerases, are used as anticancer and antiviral drugs (doxorubicin, etoposide)
• DNA damage response pathways are potential targets for cancer therapy, as cancer cells often have defects in these pathways (PARP inhibitors)
• Techniques such as DNA sequencing and PCR are used to identify mutations and study DNA replication and repair mechanisms
• Induced pluripotent stem cells (iPSCs) are generated by reprogramming somatic cells, which involves the reactivation of DNA replication and the resetting of epigenetic marks
• Gene editing technologies, such as CRISPR-Cas9, rely on the cell's DNA repair mechanisms (NHEJ and HR) to introduce targeted modifications in the genome
• Understanding the mechanisms of DNA replication and repair is crucial for the development of new therapies for genetic diseases and cancer