DNA Alkylation Flashcards
What are the methods of DNA alkylation?
Nucleotide excision repair Base excision repair Nitrogen mustards DNA methylating Agents Mitomycin C CPI PBD DNA alkylation mono/ cross linking
What is nucleotide excision repair?
DNA is damaged and damaged DNA is recognised by XPC which is stably bound to R23.
XPC-R23 and the binding of other proteins occurs, XPA and RPA are thought to facilitate specific recognition of the damage.
TFIIH unwinds the DNA duplex and this local denaturation generates a bubble in the DNA which comprise of junctions between duplex and single strand DNA.
The binding of ERCC1-XPF heterodimeric subcomplex generates a completely assembled NER multiprotein complex.
XPG is a duplex and single stranded DNA endonuclease that cuts the damaged strand at such junctions 3’ to the site of base damage. ERCC1-XPF heterodimeric protein is a duplex/ single stranded DNA endonuclease that cuts the damaged strand at such junctions 5’ to the site of the base damage.
This fragment is excised from the genome and a new section is synthesised including the damaged bases.
What are the steps for base excision repair?
N-glycosidic bond of damaged base is cleaved by DNA glycosylase leaving an abasic site in the DNA.
Sugar phosphate backbone of the abasic site is then cleaved by a bi-functional glycosylase and an AP-endoluclease. The 3’ strand break end is converted to hydroxyl allowing DNA polymerases to reinsert new bases.
Synthesis of a single base is short patch and synthesis of several bases is long patch.
What is the BER pathway involved in?
Base damage
Repair of single strand breaks
What happens when alkylation occurs in the ring nitrogen?
The base becomes unstable and may cleave from the sugar leading to single strand break.
Environmental and endogenous alkylating agents generate cytotoxic and mutagenic lesions in DNA.
How do nitrogen mustards work?
The sites of DNA alkylation include guanine N7, N3 and exo-cyclic NH2, adenine N7 and N3.
When direct ring alkylation occurs the positively charged adduct is liable to de-purinate and case strand breaks.
Inter-strand and intra-strand crosslinks on the same and between two strands of DNA.
How do DNA methylating compounds work?
The electrophile reacts with DNA and displaces the N2 as nitrogen gas and methyl is covalently attached to the DNA.
There is only one reactive center so there is no crosslinking.
How does repair on O6-methylguanine following temozolomide treatment work?
The O6-alkylguanine DNA alkyltransferase protein scans double stranded DNA for alkylation at the O6 position of guanine. Covalent transfer of the alkyl group to the conserved active-site cysteine inactivates the AGT protein and restores the guanine to normal.
What are DNA minor groove binders?
There are covalent or non-covalent types.
They are flat poly-aromatic structures which have a natural twist to fit the minor groove.
Alkylation center at the base of the minor groove and secondary non-covalent interactions are important in covalent binding.
Non covalent compounds have been modified with DNA alkylation moieties to give experimental covalent compounds.
There are many covalent compounds what have been linked to give dimeric cross linkers.
What happens in the reductive activation of mitomycin C?
The N2 and N7 atoms of guanines in the minor groove of DNA are primary alkylation sites.
Alpha attack is observed with the unnatural enantiomer of mitomycin C potentially accounting for the lower cytotoxicity.
It is completely unreactive with DNA at pH7-8 but in the presence of reductants, cross linking occurs in less than one minute.
Why can two reactions occur when DNA cross linking with dimeric CPI bizelesin?
With (+)-CC-1065 and adozelesin only a single reaction can take place but with dimeric bizelesin tow reactions can occur because of the stereochemistry in the cyclo-propyl ring only these can form inter-strand DNA duplex cross links.
How do bases form Hoogsten base pairs?
The bases must open from the Watson Crick formation and the adenine must rotate 180 degrees and reform the alternative Hoogsteen conformation.
The rate of these events is sequence dependent, some sequences rarely open and some are very prone to opening events.
What is the mechanism of action for the PBD antitumour antibiotics?
Formation of reversible aminal bond between the exo-cyclic NH2 of guanine and the C11 position of the PBD.
