What is the function of DNA-PK in NHEJ? - Answers -DNA-dependent protein kinase
-phosphorylates Ku to activate its helicase activity
-a second DNA-PK phosphorylates each other to dissociate from DNA
APE1 - Answers AP Endonuclease 1: skilled nucleic acid surgeon
Two major functions:
1. AP-site incision: DNA backbone cleavage activity at AP sites
2. 3'-5' exonuclease: proofread 3' mismatches inserted by pol beta during BER and remove 3'
DNA end damage at single strand breaks
Examples of endogenous cellular processes that damage DNA - Answers -Oxidation, alkylation,
or hydrolysis (depurination/depyrimidation) of bases
-Bulky adduct formation
-Mismatch of bases due to erros in DNA replication
Examples of exogenous sources that damage DNA - Answers -UV, X-ray
-Ionizing radiation
-Industrial chemicals
-Even oxygen
spontaneous deamination - Answers removal of an amino group generates mutagenic lesions
such as uracil, hypoxanthine, and xanthine
deamination of cytosine produces - Answers Uracil
deamination of adenine produces - Answers hypoxanthine
deamination of guanine produces - Answers xanthine
Depurination - Answers -Hydrolysis of the N-b-glycosyl bond linking a purine base to the sugar-
phosphate backbone
-elevated temperature increases the rate of depurination
Alkylation of bases occurs when: - Answers electrophilic substances seek the negative centers
in DNA, attack them and add a carbon-containing group (alkyl group)
,Emethanesulfonate (EMS) - Answers -Chemotherapeutic agent causes alkylation of guanine O6
resulting in pairing with thymine.
-DNA alylation interferes with transcription and DNA replication
-cytotoxic
oxidative DNA damage - Answers highly reactive oxidative radicals (chemical substances with
unpaired electrons) that are generated as a byproduct of cellular respiration (endogenous) or OS
(exogenous) can react with different parts of DNA structure
Guanine Oxidation - Answers -results in 8-oxoGuanine
-most common oxidative DNA base damage
-highly mutagenic
-plays a critical role in carcinogenesis
-Hoogsteen base pairing with adenine
Two forms of ionizing radiation - Answers gamma rays and x-rays
nonionizing radiation - Answers Ultraviolet rays
UV-B DNA damage - Answers cross-link adjacent pyrimidines, resulting in formation of
pyrimidine dimers which distort DNA and base-pairing and block replication
UV-A DNA damage - Answers creates mostly free radicals
Gamma and X-rays - Answers -Ionize molecules around the DNA (e.g. water)
-Cause single-strand and double-strand breaks in DNA backbone
-can alter bases (oxidative damage)
nonsense mutation - Answers A mutation that changes an amino acid codon to one of the three
stop codons, resulting in a shorter and usually nonfunctional protein.
missense mutation - Answers A point mutation in which a codon that specifies an amino acid is
mutated into a codon that specifies a different amino acid.
Mechanism of DNA polymerase proofreading - Answers If wrong nucelotide is incorporated
during DNA synthesis, polymerase pauses and transfers the 3' end of the growing strand to the
3'-5' exonuclease site where the mis-paired base is removed then then 3' end flips back to the
polymerase site and replication continues
mismatch repair - Answers Repairs base mismatches and loops (small insertion/deletions)
, Basic steps of MMR - Answers -mismatch detection
-excision
-re-synthesis by DNA polymerase
-ligation by DNA ligase
MutS - Answers -E. coli MMR protein
-Mismatch detection
-Binds to DNA mismatch and small single-stranded loops, known as sliding clamp ATPase
MutL - Answers -E. coli MMR protein
-promotes assembly of the MMR complex (MutS/MutL/ATP)
-known as molecular matchmaker
MutH - Answers -E. coli MMR protein
-endonuclease that only digests the unmethylated strand
E. coli MMR complex - Answers MutS, MutL, ATP
MutS-alpha - Answers -human MMR protein
-MSH2/MSH6
-recognizes specifically single-base substitutions and small IDLs
MutS-beta - Answers -human MMR protein
-recognizes the short ssDNA loops and larger IDLs
MutL-alpha - Answers MLH1/PMS2
-incision
MutL-beta - Answers MLH1/PMS1
-incision
Loss of MMR activity is associated with: - Answers a cancer prone disorder termed hereditary
non-polyposis colorectal cancer (HNPCC) also known as Lynch syndrome
Which two human MMR gene mutations are associated with HNPCC? - Answers -MLH1 (50%)
-MSH2 (40%)