DNA damage Flashcards
How do DNA glycosylases find damaged bases?
Scanning the genome for damaged bases Little is known about how DNA glycosylases find damaged bases in the genome. One idea, proposed on the basis of biochemical evidence and theoretical considerations, postulates the association of the glycosylase with undamaged DNA by non-specific interactions, facilitating sliding along the DNA duplex for a certain distance and scanning the sequence for irregular bases (Berg et al. 1981). Considering the structural and functional diversity of DNA glycosylases, however, there are likely to be different translocation mechanisms, variably involving features of tracking, diffusion, and hopping on the DNA (Blainey et al. 2009; Steinacher and Schär 2005; Friedman and Stivers 2010). Recently, an appealing DNA scanning concept was proposed for DNA glycosylases harboring an [4Fe4S] cluster. The underlying observation was that E. coli MutY and Nth change the oxidation state of their iron–sulfur cluster from [4Fe4S]2+ to [4Fe4S]3+ upon contact with DNA, which stabilizes the interaction. Thus, if such DNA glycosylases bind in the vicinity of each other, they might act as electron donors and acceptors for each other, making use of the DNA for charge transfer. This may facilitate the dissociation of one glycosylase upon binding of another by reduction of its [4Fe4S] cluster. If the electron transfer involved is perturbed by a base lesion between the two glycosylases, however, both will stay bound to the DNA, increasing the likelihood of damage detection (Boal et al. 2009). This way, [4Fe4S] clusters may support the search for base damage without a need of scanning the entire DNA sequence.
How many copies of OGG1 per cell
10^5
What is spontaneous hydrolysis of nucleotide residue
Loss of purines (creating apurinic sites in the DNA) has been estimated to occur 10^4 times per day in the human genome
Cytosine can slowly lose its amino group (deaminate) by hydrolysis to generate uracil, whereas 5- methylcytosine in DNA can deaminate to thymine
BER vs. NER
- Chemical modifications to DNA bases create small, non-helix distorting lesions that are repaired by the base excision repair (BER) pathway168.
○ BER involves the removal and synthesis of a single damaged base or small patch of nucleotides (2-10 nucleotides)
chemical modifications resulting in bulky lesions will distort the helix and are repaired by nucleotide excision repair (NER), which removes an oligonucleotide segment (10-24 nucleotides) surrounding the lesions.
Steps of BER
n BER, there are 4 major steps take place175:
1. Excision of the damaged base by DNA glycosylase enzymes
2. Incision of the sugar-phosphate backbone
3. DNA 5’ and 3’ end processing
4. Repair synthesis
First, repair is initiated by DNA glycosylase enzymes, which recognize the damaged base175. BER glycosylases excise the damaged base by cleaving the N-glycosylic bond between the base and the deoxyribose, typically the sugar-phosphate backbone remains intact, generating an abasic or apurinic/apyrimidinic site (AP site)174. Second, at the AP site, AP-endonuclease 1 (APE-1) cleaves the phosphodiester backbone immediately to the 5’ end, creating a ssDNA break 3′-OH and 5′-deoxyribose phosphate (5’dRP) termini, required by polymerase enzymes176. The gap is filled with the correct nucleotides by BER-specific DNA polymerase β or δ/ε174. Poly-ADP-Ribose-Polymerase (PARP) is recruited to the ssDNA break and undergoes auto-poly-ADP-ribosylation, which recruits XRCC1 and ligase I or III, forming a complex that ligates the DNA ends174
what are the two types of glycoasylases
○ Monofunctional glycosylases remove a damaged base by cleaving the N-glycosylic bond between the base and DNA backbone by hydrolysis179, creating an AP site, which is further processed by APE1 to form 3’OH and 5’dRP ends needed for polymerase function180.
○ Bifunctional glycosylases have two enzymatic activities: removing the damaged base and creating a ssDNA break.
§ Lys residue in the active site pocket, the amino group of the Lys acts as a nucleophile that attacks the carbon atom of the N-glycosidic bond, resulting in the formation of a Schiff-base intermediate, which excises the damaged base178.
§ In creating a ssDNA break, bifunctional glycoses incise the DNA 3’ to the AP site, bypassing the requirement of APE-1 for DNA end processing178. There are two types:
□ β-elimination which forms 3’-α,β-unsaturated aldehyde and a 5’-phosphate ends or
β and δ-elimination which form 3’-phosphate and 5’-phosphate ends180.
short patch vs. long patch BER
- short-patch BER.
○ A single nucleotide gap is generated in short-patch BER, and polymerase β, specific to BER, is utilized175.
○ Short patch BER is typically the dominant pathway 175.- long-patch BER
○ a gap of 2-10 nucleotides is generated, and polymerase δ/ε, along with the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), fills in the gap175.
○ Flap Endonuclease 1 (FEN1) removes the displaced single-stranded DNA flap that is created175. - The decision to carry out short-patch vs. long-patch can depend on the type of glycosylase and the psychological state of the cell62. The aforementioned glycosylase subtypes will produce biochemically different 5’ and 3’ ends at the AP site, which can have an impact on which polymerase is recruited180.
○ For example, polymerase β has a 7-fold increase in activity if there is a 5’ phosphate end compared to 5’dRP ends, in synthetic oligonucleotides181.
○ Crucially, DNA polymerase δ/ε, PCNA, and FEN1 in long-patch repair are typically expressed during the S phase of the cell cycle, such that long-patch repair is favoured in proliferating cells175.
- long-patch BER
BER in the mito
- Mitochondria were initially believed to lack DNA repair mechanisms, but decades of research have shown multiple mitochondrial DNA (mtDNA) repair pathways, including BER182. To date, there is no evidence supporting the presence of the NER pathway of bulky lesions in the mitochondria130.
- While BER in mitochondria involves similar steps as the nucleus, some key enzymes vary.
- DNA glycosylase enzymes with a mitochondrial targeting signal (MTS) can translocate into the mitochondria183.
- Variant transcripts of hOGG1 that contain an MTS include OGG1-1b, OGG1-1c, and OGG1-2a-e184.
- The endonuclease, APE1, is also present in the mitochondria for BER185.
- However, DNA synthesis in BER in the mitochondria is carried out by polymerase γ, a polymerase which functions exclusively in the mitochondria186.
- The final ligation step is performed by Ligase III, without the scaffolding protein XRCC1, which is absent in the mitochondria187.
Importantly, DNA repair in the mitochondria can be overwhelmed, leading to mtDNA destruction either through direct degradation or mitophagy, a form of autophagy specific to the mitocondria188.
repair of 8-oxoG in the nucleotide pool
- ROS can oxidize GTP to 8-oxo-GTP in the nucleotide pool4.
The MutT homolog enzymes (MTH1, MTH2, MTH3 or Nudix-type 5 [NUDT5]) are responsible for the hydrolysis of 8-oxo-GTP back to GTP in the cytosol and nucleus189.
