DNA's built-in protection against errors (2)

complementary strands make repairs easy, DNA polymerase proof-reads

Exogenous causes of DNA damage

UV light

Radiation

man-made agents (chemo)

heat

viruses

benzo(a)pyrenes (burnt meat)

tobacco byproducts

Endogenous causes of DNA damage

internal cellular damaging agents produced by the cell

ROS (reactive oxygen species) aka free radicals

nitric oxide

Senescence

cell becomes "dormant" (stops replicating) in response to irreparable DNA damage

Apoptosis

programmed cell death in response to irreparable DNA damage

DNA damage that can be repaired (4)

covalent modification of bases (methylation, alkylation etc)

'mismatches' of the normal bases as a result of proofreading failure

breaks in DNA backbone (apurination, apyrimidination)

formation of covalent crosslinks between bases (ex: cyclobutane dimer between adjacent T-T)

mutagens

induce reactions that modify bases or cause base changes during subsequent DNA replication

oxidative deamination

converts C to U

apurination or apyrimidination

loss of purine of pyrimidine base

O⁶-methyl guanosine

alkylation of guanine (chemoptherapeutic agents are often alkylating agents) causes it to pair with T rather than C

cyclobutane dimer

forms between adjacent T-T, blocking DNA polymerase

formed by UV radiation

Cytosine deamination

C deaminated to U, replication-->A, second replication-->T

overall, C-->T

Single-strand breaks

result of problem with ligation of Okazaki frags

Double strand breaks

result of errors in recombination or ionizing radiation

Types of DNA repair (3)

Excision repair

Direct repair (direct demethylation)

Repair of Double-Stranded Breaks (DSBs)

BER

Base Excision Repair

removal of a single base used to correct DNA that's been depurinated, deaminated or alkylated

1)DNA glycosylases recognize and remove damaged base by cleaving base/sugar bond

2)apurinic/apyrimidinic endonuclease (APE) and a phosphodiesterase remove sugar-phosphate residue

3)DNA polymerase β fills the gap

4)DNA ligase seals the backbone

problem with Uracil substitutions

NOT generation of a mutation bc rounds of replication will fix, rather danger is if U is subbed within a regulatory region (alters transcription)

DNA glycosylase

BER enzyme that 'patrols,' looking for damaged bases

UNG

specific for removal of U opposite of T when dUTP has inappropriately been used in DNA synthesis

hSMUG1

removes U from single stranded DNA (recognizes it in transcription bubble), U present often a result of C deamination

TDG and MBD4

TDG and MBD4: remove U and T when opposite G residues

NER

nucleotide excision repair

used primarily for removal of pyrimidine-dimers and other lesions which distort DNA helix

recognizes abnormal kinks/bulges in DNA

BER vs NER

NER removes not onl the abnormal nucleotide but several bases (24-32) around it

xeroderma pigmentosum

genetic disease caused by mutation in NER gene

photosensitive, susceptible to skin cancers

genes involved in NER

XPA through XPG (named for xeroderma pigmentosum)

some named for UV that causes damage (ex: UVrD--helicase unwinds DNA during NER)

Steps of NER

Recognition

Incision

DNA synthesis

Ligation

importance of NER

rate of NER directly proportional to lifespan of organism

Transcription-coupled NER

NER applied to UV-damaged, transcriptionally-active genes

activated when lesion blocks transcription, stall in RNA polymerase II induces NER

Mismatch repair

excision repair that removes mistakes in DNA replication (mismatches cause distortion in double helix)

problem=recognize which base is the mismatch

solution=use methylated DNA strand to recognize older parental strand

Lynch syndrome

hereditary colon cancer (autosomal dominant)

Amsterdam Criteria

proteins involved in mismatch repair are missing, therefore mm repair non-functional-->cancer

Direct repair

direct demethylation with MGMT (O⁶-methylguanine-DNA-methyltransferase)

Adj C-G often methylated (after DNA rep, as mech to inactivate genes OR by carcinogens/endogenous methyl carriers like S-adenosyl methionine)

MGMT recognizes/demethylates O⁶-methyl guanine (v mutagenic--base pairs with T instead of C)

MGMT

O⁶-methylguanine-DNA-methyltransferase

photoreactivation

primarily found in unicellular organisms

thymidine dimers removed by photolyase, enz binds dimer, absorbs light and breaks bonds

repair of DSBs

occurs during recomb, meiosis or afterexposure to ionizing radiation

no detection: cell death

improper/incomplete repair: tumorigenic

Homologous recombination repair

HRR

DSB repair used after S₁ phase, kinases are activated when break is detected--slow cell cycle

info from sister chromatid used to fix broken strand

Non-homologous end joining

NHEJ

used during G1 phase, less accurate bc no alignment w sister chromatid

proteins recognize and bind to broken ends

trimming of overhangs results in deletion of a few nucleotides

Ataxia-Telangiectasia

defects in repair of DSBs

hypersensitive to ionizing radiation, susceptible to cancer(defective A-T gene)--50% inc in odds of BrCa in females, chromosomal instability, abnormalities in gen. recomb., defective signalling to apoptosis

BRCA1 & BRCA2

DNA repair genes originally detected in breast cancer, now known to contribute to many types of cancer