DNA Repair and Genome Integrity 1/2

Lecture 8

Why is DNA subject to constant change?

it is getting damaged constantly because of all the reactions in the cell

exampled of the constant change to which DNA is subjected

• hydrytic depurinations (water interactions in the genome)

  • 2000-100000 /day/cell

• cytosune deaminations

  • 1/5days/cell

• guadinine oxidaion

  • 1/5days/cell

• methylation of adenine

• 600Lday/cell

=> these account for a lot of change and DNA repari potential because there ares estimated 3.72×10^13 cells in our body

what are three processes that produce modified bases? and what can the modification of bases do ?

• deamination

  • when amine group is removed => chemistry of base is changed

• oxidation

• alkylation

  • small molecules containing carbon are attacehd to these bases (i.e. metjhylation)

• this can alter the original base pairring 

• may block the activity of the DNA polylerases during DNA replication

  • so have to be reparied for DNA replication to occur

mutations

permanent, transmissible, changed to the genetic material of a cell or organism

• can occur spontaneously 

• can occur by transposable elements (jumoing genes)

• can occur because of errors during replication

mutagens

chemical compounds like UV radiation or ionizing radiation that increase the frequency of mutations

carcinogen

an agent that causes cancer

• many carcinogens are mutagens

Where do Mismatches come from?

DNA polymerase can introduce errors

• alone introduces 1 error every 10000 nucleotides

what are the repair mechanisms for mismatch errors?

• proofreading exonuclease

  • an activity of DNA polymerases to correct the mistakes they made

  • activity increases fidelity by 100 fold

• MMR: Mismatch repair 

  • further increases fidelity by 1000 fold

adding this up: probability of incorporating a single mistake of 10^9 or 10^10 per nucleotide in the human cell

why does the n° of mutations fathers ingerit to their offspring increase with age?

because of continuous production of germ cells

what can we say about the rate of mutations in viruses vs cells

It is much higher in viruses

• for higher probability of selecting more successful variants

what DNA polymerases have a 3’ to 5’ exonuclease activity?

DNA polymerase epsilon

DNA polymerase delta

(not DNA pol alpha!!)

where does MMR often act in proximity to?

the replisome

with what protein from the replisome do some of the proteins from the MMR associate with

PCNA

(the proteins that keep the polymerases on DNA)

Mechanism of MMR in humans

(1)

• error incorporated into newly synethizes strand

• => MSH2 and MSH6 bind to daughter strand (these proteins detect the mistake)

(2)

• => triggers binding and activity of MLH1 endocunclease (dimerized with PMS2), which make cuts near the mismatch.

  • MLH1 starts from inside (why “endo”) to make cut near the mismatch to mark it

  • the cuts flank the mutaiton

• Then, DNA helicase unwinds and DNA exonuclease digests segment of the daughter strand

  • this helicase is different from the one used during DNA replication

(3)

• Gap repair by Pol delta and DNA ligase

  • aka a single strand gap 

  • nb. Pol delta is the lagging strand polymerase 

What spontaneously occurs forming cytosine into uracil?

deamination

• amine group is removed by hydrolysis (reaction with water) and the NH2 amine group is replaced with an oxygen 

• implication: C pairs with G wherease U pairs with A

  • error needs to be caught in time in order to not lead to bad pairing

Base Excision Repair (BER)

repairs small non-helix distorting lesions such as those from oxidation, deamination and alkylation

Mechanism of Base Excision Repair (BER)

(1)

• DNA glycolase

  • recognizes the innapropriate or damaged DNA

  • hydrolyzes the bond between the mispaired base and the sugar phosphate backbone

• after the removal of the base there is a gap

(2)

• Apurinic/apyrimidinic endonuclease 1 (APE1) cuts the DNA backbone

  • at the site where ther is no base

(3)

• AP lyase associated with DNA polymerase beta removes the deoxyribose phosphate

  • DNA pol beta is specific for repair

(4)

• DNA polymerase beta fills the gap by acting like a polymerase and DNA ligase seals the nick in the sugar-phosphate backbone

nb. this system relies on double stranded DNA

DNA glycolase

• enzyme that performs the first step in BER by recognizing the and removing the damaged or inappropriate DNA bases from the DNA molecule

• “glycolase” beause breaks the covalent bond of the base to the ribose (which is a sugar)

• It provides specificity in repair — only removes the bases that need to be removed 

  • there are different types of DNA glycolases in our cells

    • the one that removes uracil is called UNG

Bulky Lesions

modifications so big that they can change the regular double-stranded structure of DNA 

• often caused by photon of UV light

• These lesions disrupt the normal double helix structure, blocking DNA replication and transcription

• Often repaired by a system called nucleotide excision repair (NER)

example of a bulky lesion: Thymine-thymine dimer (aka a pyrimidine dimer)

• the two covalently linked thymines T-T form a rigid shape that distorts the DNA helix

• like other bulky lesions, disrupts DNA replication and transcription

Bulky lesion example: from food contaminated by fungi Aspergillus or smoke from organic material (like cigarette or charboiled food)

• Ingestion of Aflatoxin B

  • reacts with DNA and forms a covalent bond

  • enzymatic modification

  • spontaneous reaction with N7 of guanosine

• benzo(a)pyrene (from the smoke of organic material) will generate modification similarly to Aspergillus to DNA 

Nucleotide Excision Repair (NER) mechanism (for bulky lesions)

(1) initial damage recognition

• A complex formed by proteins 23B and XP-C do the initial recognition of the DNA lesions

(2) opening of the DNA double helix

• The complex of 23B and XP-C recruits the transcription factor TFIIH

  • TFIIH catalyzes the unwinding of the DNA region around the lesion

    • This recruits RPA and XP-G (which also have unwinding activity)

      • (nb. these are also used in DNA replication — pattern that proteins being used for replication also used for repair )

(3) XP-F and XP-G endonucleases

• XP-F recruited

• both XP-F and XP-G have endonuclease activities (aka they cut DNA)

  • generate cuts 24-32bp apart — a bigger gap is generated

    • releasing fragment carrying the lesion which will be degraded

(4) DNA polymerase & DNA ligase

• the ssDNA gap generated is filled by DNA polymerase (probably Pol delta)

• then joined to generate continuous strand by DNA ligase

nb. this system relies on double stranded DNA

What are example of humans Hereditary diseases and cancers associated with DNA Repair Defects

• Hereditary nonpolyposis colorectal cancer

  • DNA mismatch repair affected

• Xeroderma Pgmentosum

  • NER

what happens is a bulky lesion or other type of lesion is not repaired in time and the DNA is replicated

• BER and NER repair mechanisms rely on double stranded DNA

• if not caught in time the DNA polymerases will stop (stalled by replisome)

  • when replisome encounters DNA lesions that weren’t previously repaired the DNA polymerase of the replisome stops

    • this prevents the completion of DNA replication

• big issue for cell because it dies 

what repair mechanism is used as a last resort

Translesion Synthesis is a last resort solution for when replication

• special type of DNA polymerase used: translesion(TLS) polymerases

what is special about translesion (TLS) polymerases

• they are able to bypass the lesion using the damaged DNA as a template

  • however, often use the incorrect based at position opposite the lesion (because they have a single strand they don’t know the opposite base)

  • do not have proofreading exonuclease activity

    • hence they are error prone

what is the implication of using TLS polymerases as a last resort (trade-off)

negative

• easily make mistakes

  • leads to mutations

    • often at a great cost in humans

positive

• mutations can be halful in bacteria