DNA Replication Overview

DNA Overview

• DNA consists of nucleotides linked by 3'-5' phosphodiester bonds.
• DNA encodes specific gene products (proteins) through its unique sequence.
• Sequences are read in the 3' to 5' direction.
• Replication produces a complementary copy in the 5' to 3' direction.

Steps in Prokaryotic DNA Synthesis

• DNA replication is semi-conservative; each new DNA molecule has one original strand and one new strand.

Separation of Complementary DNA Strands

• In prokaryotes, replication starts at a single origin of replication, a unique nucleotide sequence.
• In eukaryotes, replication starts at multiple sites with consensus sequences rich in AT bases.

Replication Fork Formation

• DNA strands unwind at the replication fork, creating a "V" shape.
• Replication is bidirectional, with the replication fork moving in both directions from the origin.

Prokaryotic Replication

• Replication is semiconservative, producing a new strand and conserving one strand from the parent DNA.
• Initiation begins at specific nucleotide sequences rich in AT.
• A prepriming complex is required, containing:
• dna A protein
• DNA helicases
• Single-stranded DNA-binding (SSB) proteins
• Topoisomerases I and II unwind supercoils.

Topoisomerases

• Topoisomerases relieve supercoils formed during DNA strand separation.
• There are two types of topoisomerases: I and II

Topoisomerase I and II Inhibitors

• Etoposide inhibits human topoisomerase II and is used as an anticancer agent.
• Ciprofloxacin inhibits bacterial DNA gyrase, and is used to treat infections.

Elongation Steps

• DNA polymerase III reads both parental strands in the 3'-5' direction.
• The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously.
• RNA primer (~10 nucleotides of single-stranded RNA) forms a short hybrid sequence with DNA, providing a 3'-OH for the first d-NTP (primase).
• DNA replication stops if any of the d-NTPs are low or absent.
• DNA polymerase III is processive, bound to the template strand by the β subunit.
• DNA polymerase III has 5'-3' polymerase activity and 3'-5' proofreading exonuclease activity.
• DNA polymerase I has 5'-3' polymerase activity, and 3'-5' and 5'-3' exonuclease activity to remove the RNA primer.
• Okazaki segments on the lagging strand are made because it discontinuously synthesised.
• Gaps between Okazaki segments are filled and RNA primers are replaced with DNA.
• Proofreading is performed by 3'-5' exonuclease activity.
• Ligase joins Okazaki segments by forming phosphodiester bonds.

Key Concepts in Replication

• Helicase facilitates local separation of strands at the replication fork.
• Single-strand DNA-binding proteins stabilize single strands.
• RNA primer building on newly separated strand is synthesised by primase.

Exonucleases and Proofreading

• DNA polymerase has proofreading function to excise mismatched nucleotides.

Chemotherapeutic Agents

• Sulfonamides and methotrexate inhibit purine synthesis, thereby inhibiting DNA replication.
• Sulfonamides are used in bacteria
• Methotrexate is used in humans

Eukaryotic Replication

• The cell cycle includes phases Go, G1, S, G2, and M; postmitotic cells do not divide.
• There are five classes of DNA polymerase.
• DNA polymerase ɑ (pol III) has primase activity to start primer formation.
• DNA polymerase δ elongates leading and lagging strands and has 3'-5' exonuclease activity.
• DNA polymerase β,ε is used for repair.
• DNA polymerase γ carries out mitochondrial replication.
• Telomeres are single-stranded T and G repeats complexed with proteins, replacing sequences vacated by RNA primers at lagging ends.
• Telomerase, present in germ and tumor cells, has reverse transcriptase activity.

Viral Reverse Transcriptase

• Retroviruses' genome is encoded by RNA.
• Retroviruses use reverse transcriptase (RT) to build viral DNA from their RNA template.
• Viral DNA integrates into host DNA and replicates when host DNA replicates.
• Inhibitors of viral replication include RT inhibitors, DNA integrase, and protease inhibitors.
• Nucleotide analogues such as Ara C, dideoxyinosine, and AZT can terminate DNA synthesis, acting as antiviral or chemotherapy agents.

DNA Repair

• Thousands of mutations occur daily due to environmental factors like UV light and nitrous acid.

Types of Mutations

• Point mutations
• Insertions
• Deletions

Mechanisms of DNA Repair

• Mismatch repair: endonucleases remove mismatched bases.
• UV light damage: repair of thymine dimers by a UV-specific endonuclease; defects cause xeroderma pigmentosum and increased cancer risk.
• Correction of altered bases: removes altered bases from deamination (nitrates) or spontaneous loss of bases (glycosylases).

Methyl-Directed Mismatch Repair (NER)

• Mut proteins recognize mismatch and identify the methylated parent strand.
• Polymerase fills created gap, ligase joins the newly synthesised DNA piece to the original strand.

Excision Repair of Pyrimidine Dimers in DNA (NER)

• UV-specific endonuclease excises pyrimidine dimers.
• Polymerase and ligase used to repair the created gap.

Base Excision Repair

• Uracil-N-glycosylase removes damaged base.
• DNA polymerase and DNA ligase used to repair created gap.