08 - DNA Replication & Repair

3’ end

5’ to 3’

dNTPs are substrates, and the α phosphate on the dNTP is retained on the strand with the β and γ phosphates being released as pyrophosphate; the hydrolysis of pyrophosphate to phosphate drives the rxn forward

Most of the nucleoside analogs used as drugs lack a 3’-OH group, and therefore act as chain terminators

It is a specific DNA sequence which is AT rich; there is one in prokaryotes and several in eukaryotes

Protein-DNA and protein-protein interactions

• Catalyzes unwinding of the DNA strand; it is unidirectional and energy-dependent; multiple helicases are present in prokaryotes &
• eukaryotes

ssDNA-binding proteins bind ssDNA rightly and shift the equilibrium between ssDNA and dsDNA towards ssDNA; it prevents nucleases from attacking the ssDNA

DNA topoisomerases introduce swivel points along the double helix; type1 makes single strand nicks and relaxes one supercoil at a time; type2 cuts both strand of DNA and relaxes two supercoils at a time; both topoisomers are ATP dependent

In anti-cancer drugs they are targeted to stop the process of replication, generating single or double strand breaks in the DNA; in anti-bacterial drugs they inhibit gyrase to stop replication & transcription, and generate double strand breaks in DNA

• Since DNA pol cannot initiate the synthesis of a new strand, it can only catalyze the addition of a nucleotide to the 3’-OH group of a growing
• strand, RNA primer provides the initial free 3’-OH group

Primer, template, Mg²⁺/Zn²⁺ and all four dNTPs

Proofreads the newly synthesized DNA strand; if an incorrect nucleotide is incorporated then DNA synthesis is paused b/c of incorrect positioning of 3’-OH; the last 2-4 bps unwind, and the unwound region of primer binds to the 3’ to 5’ exonuclease site; the incorrect nucleotide is removed, and the primer-template junction is reformed and rebind to the synthetase site

Degrades RNA or DNA strands that are hybridized to template DNA strands; in prokaryotes this removes RNA primers

Present in enzymes with high processivity; it works as a sliding clamp, encircling the dsDNA to ensure that polymerase stays at the primer-template junction and is responsible for the processivity of the polymerase enzyme

Found in prokaryotes; has low processivity, adding only ~50 nucleotides/min/enzyme molecule; it is mostly involved in RNA primer removal and DNA repair

Found in prokaryotes; lacks proof reading activity, however is involved in DNA repair

Found in prokaryotes; has high processivity, adding ~9000 nucleotides/min/enzyme molecule; replicates most of the DNA

Found in prokaryotes; involved in DNA repair

Complex, found in prokaryotes, comprised of a helicase, ssDNA-binding proteins, primase and dimeric Pol III holoenzyme; this catalyzes the synthesis of both strands of DNA

The removal of the RNA primer is mediated by DNA Pol I, and 5’ to 3’ exonuclease removes NTPs from the 5’ end; the synthetase activity adds dNTPs to the 3’ end of the previous strand; the okazaki fragments are sealed by DNA ligase and 2 circular DNA molecules linked together in a chain is untangled by topoisomerase

Found in eukaryotes; tightly interacts with primase

Found in eukaryotes; functions in repair

Found in eukaryotes; mitochondrial polymerase

Found in eukaryotes; polymerase of the lagging strand; has proofreading activity (3’ to 5’ exonuclease activity)

Found in eukaryotes; polymerase of the leading strand; has proofreading (3’ to 5’ exonuclease) activity

Removes RNA primers in eukaryotic cells

Replication is initiated by Pol α/primase comples; after Pol α incorporates a few nucleotides, Pol δ or Pol ε replaces Pol α

Replication errors; spontaneous alteration in the chemistry of DNA bases [cytosines deaminate to uracil, and 5-metylcytosines deaminate to thymine (these are hotspots for mutations)]; transposons

Source of DNA damage; alkyl groups are covalently attached to bases

Source of DNA damage; inter-strand crosslinking by covalent linkage

Source of DNA damage; substitute for normal bases

Source of DNA damage; intercalate between bps and cause insertion or deletion mutations

Proteins recognize deformity in DNA molecule; recruits endonuclease and other pathway-specific enzymes; exonucleases fix problem

Involved in the removal of alkyl groups attached to the O⁶ in guanine; mediated by O⁶-methylguanine methyl transferase (MGMT) or alylguanine alkyl transferase (AGAT)

• Repair of replication errors and small insertion or deletion; Mut proteins are involved in the
• recognition of the mismatch, make a nick in the newly synthesized strand; defects in the MMR pathway can lead to sporadic cancers

Previously called hereditary non-polyposis colon cancer (HNPCC) commonly has mutation in the Mut protein homologs

Repair of damaged bases or ssDNA breaks; generally works by removal of the base by a glycosylase, cleaving the sugar-phosphate backbone by an AP endonuclease which removes the abasic nucleotide, insertion of the correct nucleotide by polymerase, and ligation of the backbone by ligase

Repairs a variety of helix-distorting DNA lesions such as pyrimidine dimer, bulky adduct of base, cisplatin-induced DNA damage; after defect recognition, nucleotides are removed by pathway-specific exonuclease and helicase and the gap is filled in by DNA polymerase; also called the global NER pathway because it scans the entire genome for mutations

Results for a defect in the global NER pathway; first sign is usually excessive, long-lasting sunburn, dry skin, excessive freckling, eye damage, and keratosis; has a 2000-fold higher rate of fatal skin cancer

Results from defect involved in transcription-coupled NER pathway; RNA synthesis following exposure to UV is drastically reduced; results in dwarfism and developmental delay

HR repairs the dsDNA break by using a duplicate set of DNA as the template; this is a very precise pathway; this is particularly active during the late S or early G2 phase of the cell cycle; BRCA1 and BRCA2 are involved in the stabilization of the breaks and promoting HR over NHEJ

NHEJ repairs the dsDNA break directly w/o any outside information; the proteins involved in the pathway bind the free ends of the broken DNA, trim the ends and ligate them back together; this may result in the addition or removal of a few nucleotides at the repair site; used to repair most of the double strand breaks, particularly during the G1 phase