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DNA repair Flashcards

1
Q

Why is DNA repaired?

A

DNA is the only macromolecule that is repaired. A diploid cell has only two copies of each genomic DNA but hundreds to thousands of copies of each RNA and protein molecules. Damaged proteins and RNAs can be quickly disposed and replaced using the information encoded in the DNA, but DNA molecules themselves are irreplaceable. Maintaining the integrity of the information in DNA is a cellular imperative, supported by elaborate DNA repair systems, with over 100 genes required for DNA repair in very small genomes, and many more in humans.

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2
Q

Mutations

A

Changes in DNA molecules can cause mutations. After replication, these changes result in a permanent alteration of the base sequences in the daughter DNA. Cancer and many human diseases are the consequence of mutation due to DNA damage and inadequate DNA repair. Mutations can also be advantageous, as they are responsible for evolution and allelic variation or polymorphisms in a population that makes us each unique. If mutations occur in the germline, they can be inherited by the offspring.

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3
Q

Change-causing mutations include:

A

1)Uncorrected errors made during DNA replication and 2) Damage that occurs to replicating or nonreplicating DNA, such as oxidative damage caused by products of normal metabolic activity of the cell, cleavage of a DNA strand caused by radiation and chemicals, chemical alterations to the base (e.g. alkylation), loss of a base (depurination / depyrimidination), loss of an amine group of the base (deamination), sunlight induced thymine-dimers.

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4
Q

what is the product of the deamination of C ?

A

yields a U, if the U is not replaced with a C via DNA repair, then during DNA replication an A will be incorporated opposite the U, and the U will then be replaced with a T. The end result is mutating a CG base pair permanently to a TA base pair.

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5
Q

How do mutations lead to cancer?

A

A key example of the profound importance of DNA repair is seen in cancer. If DNA damage is not properly repaired, the result is mutations. If mutations occur in genes encoding components of the DNA repair machineries or DNA damage sensing and signaling pathways the result is more mutations and genomic instability - accumulation of changes to the DNA. If there are mutations in these pathways such that the cell cannot kill itself or stop dividing however, the result is increased risk of developing cancer. Clearly DNA damage and repair is essential for keeping cancer in check. clinicians will also use DNA damage, via radiation therapy and chemotherapy, to treat cancer patients - by killing the cancer cells.

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6
Q

point mutations

A

substitution of one base for another

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7
Q

insertions mutations

A

addition of one or more nucleotides within a DNA sequence

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8
Q

deletions mutations

A

removal of one or more nucleotides from a DNA sequence

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9
Q

Spontaneous base loss

A

includes depurination and depyrimidination

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10
Q

Spontaneous deamination of cytosine

A

the hydrolysis reaction of cytosine into uracil (a point mutation), releasing ammonia in the process. This is corrected for by the removal of uracil base excision repair

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11
Q

Spontaneous deamination of 5-methylcytosine

A

results in thymine and ammonia. This is corrected for by the removal of thymine base excision repair

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12
Q

Deamination of guanine

A

results in the formation of xanthine. Xanthine, in a manner analogous to the enol tautomer of guanine, selectively base pairs with thymine instead of cytosine. This results in a post-replicative transition mutation, where the original G-C base pair transforms into an A-T base pair. Correction of this mutation involves the use of base excision repair

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13
Q

thymidine dimers

A

A pair of abnormally chemically bonded adjacent thymine bases in DNA, resulting from damage by ultra-violet irradiation. dimers interfere with base pairing during DNA replication, leading to mutations. Can be repaired with direct reversal, nucleotide excision repair,

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14
Q

Base alkylation

A

the addition of alkyl (methyl, ethyl, occasionally propyl) groups to the bases or backbone of DNA. Alkylation can occur through reaction of compounds such as S-adenosyl methionine with DNA. Alkylated bases may be subject to spontaneous breakdown or mispairing (a point mutation). Repaired with direct reversal, base excision repair, and mismatch repair

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15
Q

Base oxidation

A

Oxidative damages to bases are caused by reactive oxygen species (ROS) that are generated during cell metabolism. the process of oxidative damage on Deoxyribonucleic Acid. It occurs most readily at guanine residues due to the high oxidation potential of this base relative to cytosine, thymine, and adenine (e.g. guanine-> 8oxoG). It is widely believed to be linked to certain disease and cancers. It blocks DNA replication and is bypassed by regular polymerase and mispairs with A. Repaired with base excision repair and mismatch repair

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16
Q

8-Oxo-2’-deoxyguanosine (8-oxo-dG)

A

an oxidized derivative of deoxyguanosine. 8-oxo-dG is one of the major products of DNA oxidation. Concentrations of 8-oxo-dG within a cell are a measurement of oxidative stress.

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17
Q

What can cause bulges in DNA?

A

insertion/ deletion of nucleotides, bulky chemical adducts, replication errors (mismatch), intra/ inter-strand crossslinks, they interfere with replication and transcription

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18
Q

insertion/deletion of nucleotides

A

a molecular biology term for the insertion or the deletion of bases in the DNA of an organism. Repaired by nucleotide excision repair

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19
Q

bulky chemical adducts

A

When a chemical binds to DNA, the DNA becomes damaged, and proper and complete replication cannot occur to make the normal intended cell. This could be the start of a mutation, or mutagenesis, and, without proper DNA repair (DNA repair happens naturally under normal circumstances), this can lead to carcinogenesis, the beginnings of cancer. Can be repaired by nucleotide excision repair

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20
Q

replication errors (mismatch)

A

repaired with mismatch repair

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21
Q

intra/ inter- strand crosslinks

A

crosslinking of DNA occurs when various exogenous or endogenous agents react with two different positions in the DNA. This can either occur in the same strand (intrastrand crosslink) or in the opposite strands of the DNA (interstrand crosslink). Crosslinks also occur between DNA and protein. DNA replication is blocked by crosslinks, which causes replication arrest and cell death if the crosslink is not repaired. Repaired with nucleotide excision repair, and single strand base repair

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22
Q

DNA strand breaks

A

both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. Repaired with direct reversal, single and double strand break repair

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23
Q

Stalled DNA replication fork

A

Some chemicals can cause RF-stalling DNA damage that leads to diseases such as cancer. Can be repaired with double strand break repair

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24
Q

Benzo(a)pyrene

A

found in coal tar with the formula C20H12. Its metabolites are mutagenic and highly carcinogenic (BPDE) BPDE adduct causes insertion of A opposite of G by DNA polymerase. This disrupts the normal process of copying DNA and induces mutations, which explains the occurrence of cancer after exposure. Changes GC to TA

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25
Q

O6-meG

A

produced with base alkylation of G causing mismatch with T which is then matched with A causing a point mutation

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26
Q

Direct reversal of the damage

A

Reversal of a specific type of single-stranded DNA break by DNA ligase. Reversal of UV-caused base damage (T-T T-C dimers) by photolyase. Reversal of base alkylation by O6-meG methyltransferase (MGMT).

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27
Q

O6-alkylguanine DNA alkyltransferase (MGMT)

A

It repairs the naturally occurring mutagenic DNA lesion O6-methylguanine back to guanine and prevents mismatch and errors during DNA replication and transcription by removing the methyl group from O6-methylguanine . loss of MGMT increases the carcinogenic risk in mice after exposure to alkylating agents. It is evolutionarily conserved
and a classical example of “direct reversal” type of DNA repair. Tumor-associated mutations in MGMT reduces its DNA repair activity. MGMT is silenced via promoter methylation in ~45% of human glioblastomas.

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28
Q

Excision of damaged, mispaired, or incorrect bases

A

mechanisms include base excision reapir, nucleotide excision repair, and mismatch repair. all three repair machineries take advantage of the double-stranded nature of the DNA molecule to copy the correct information from the intact strand of DNA to the damaged strand.

29
Q

Base excision repair (BER)

A

repairs base damages that do not distort the DNA. It can repair: Oxidized bases (8-oxoguanine), Alkylated bases, Deaminated bases (Xanthine formed from deamination of guanine, Thymidine products following deamination of 5-methylcytosine are more difficult to recognize, but can be repaired by mismatch-specific glycosylases, Uracil inappropriately incorporated in DNA or formed by deamination of cytosine)

30
Q

Nucleotide excision repair (NER)

A

repairs base damages that distort the DNA. (thymine dimers)

31
Q

Mismatch repair (MMR)

A

removes misincorporated nucleotides during DNA replication. fixes errors in nucleotide incorporation made by DNA polymerase during DNA replication. The mismatched base pair is recognized shortly after DNA synthesis by the MutS and MutL proteins in bacteria; their mammalian counterparts are MSH (MutS Homolog) and MLH (MutL Homolog) proteins. In bacteria, the newly synthesized strand of DNA is identified by the MMR machinery because it is not yet methylated. An endonuclease cleaves the phosphodiester backbone of the new strand of DNA. An exonuclease chews away the new DNA strand including the mismatch nucleotide while a helicase assists with the unwinding of the double helix. DNA polymerase repairs the resulting single strand gap by incorporating complimentary base pairs, and DNA ligase seals the phosphodiester backbone.

32
Q

Tolerance/Bypass of base damage

A

trans-lesion synthesis, It involves switching out regular DNA polymerases for specialized translesion polymerases, often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides. Such DNA polymerases will insert AAA’s because this mechanism is mostly used with T-T dimers

33
Q

Strand break repair of damage to the DNA backbone

A

includes single strand break repair and double stranded break repair

34
Q

Double-strand break repair (DSBR)

A

Includes homologous recombination and nonhomologous end- joining

35
Q

Direct reversal of the damage

A

some examples include ligation of a break in the phosphodiester backbone of the DNA by DNA ligase and repair of O6-methylguanosine by O6-methylguanosine methyltransferase (MGMT).

36
Q

Excision Repair

A

excision of the damaged region, followed by precise replacement. In general, excision repair involves removal of the segment of DNA strand that contains a damaged region or mismatched bases, filling in the gap by the action of a DNA polymerase that uses the undamaged sister strand as a template, and ligation of the newly synthesized segment to the remainder of the chain. Basically all excision repair pathways require endonuclease and/or exonuclease, DNA polymerase and DNA ligase. There are three major types of excision repair: NER, BER and MMR; each pathway uses specific enzymes.

37
Q

Steps common to all three excision repair mechanisms

A

1) Recognition of the damaged/mismatched nucleotide. 2) Endonuclease-mediated cutting of the phosphodiester backbone flanking the damaged/mismatched nucleotide. 3) Nuclease-mediated removal of the DNA fragment containing the damaged/mismatched nucleotide. 4) DNA polymerase-mediated synthesis of the missing nucleotides by copying nucleotide sequence from the intact DNA strand. 5) DNA ligase-mediated sealing of the remaining nick in the phosphodiester backbone.

38
Q

Base excision repair (BER)

A

removes DNA lesions that are missed by the NER process, but do not necessarily block polymerase function or distort the DNA structure. BER requires a family of enzymes called glycosylases, each recognizing a specific type of altered base. For example, uracil glycosylase recognizes uracils in DNA that result from cytosine deamination, and 5- methylcytosine-DNA glycosylase recognizes 5-methylcytosine to initiate DNA demethylation. The glycosylase flips the altered base out from the double strand helix and then hydrolyzes the N-glycosidic bond (the bond within the nucleoside between the base and the sugar) to remove the damaged base, producing an apurinic or apyrimidinic (AP site) site. The AP site is then removed by an AP-specific endonuclease and an AP lyase, and the resulting gap is filled by a DNA polymerase and the nick sealed by a DNA ligase.

39
Q

glycosylases

A

a family of enzymes involved in base excision repair. It catalyze the first step of this process. They remove the damaged nitrogenous base while leaving the sugar-phosphate backbone intact, creating an apurinic/apyrimidinic site, commonly referred to as an AP site. This is accomplished by flipping the damaged base out of the double helix followed by cleavage of the N-glycosidic bond. Each glycosylase recognize only a particular base damage, a particular inappropriate base, or a particular mispairing.

40
Q

Global genome NER

A

recognizes distorting DNA lesions in any region of the genome. Following recognition of the distorting DNA lesion by proteins unique to each of the two pathways, the remainder of the NER occurs in the same manner.

41
Q

Transcription-coupled NER

A

recognizes distorting DNA lesions in regions that are actively transcribed. Following recognition of the distorting DNA lesion by proteins unique to each of the two pathways, the remainder of the NER occurs in the same manner.

42
Q

Nucleotide excision repair (NER)

A

removes DNA lesions that distort the DNA structure and block RNA or DNA polymerase movement on the DNA. Examples of these types of lesions are thymine dimers resulted from exposure to UV and bulky DNA adducts caused by exposure to carcinogens. The distorted DNA is recognized by a multi-protein complex that contains endonuclease activities, which cut on strand of the phosphodiester backbone on both sides of the DNA lesion and generate an oligonucleotide containing the damaged nucleotide. A DNA helicase then unwinds the DNA and releases the single-strand oligonucleotide that includes the DNA lesion, which is then chewed away by an exonuclease. DNA polymerase comes in and inserts the nucleotides that are complimentary to the remaining, intact DNA strand. DNA ligase seals the gap in the phosphodiester backbone to complete the repair process. The lesion is either recognized by global genome NER and transcription- coupled NER. Is more verstille than BER

43
Q

steps of base excision repair

A

removes base damage that doesn’t distort the DNA duplex by 1) Modified base is recognized by a specific DNA glycosylase, which hydrolyzes the N-glycosidic bond, yielding an AP site. 2) An AP site-specific endonuclease (APE1) cleaves the sugar-phosphate backbone 5’ to the AP site. 3) Another endonuclease cuts 3’ to the AP site,
removing the deoxyribose phosphate. 4) The resulting gap is filled by DNA polymerase, and the nick sealed by DNA ligase.

44
Q

AP site

A

apurinic/apyrimidinic site, a location in DNA (also in RNA but much less likely) that has neither a purine nor a pyrimidine base, either spontaneously or due to DNA damage.

45
Q

APE1

A

Apurinic/apyrimidinic (AP) endonuclease, an enzyme that is involved in the DNA base excision repair pathway (BER). Its main role in the repair of damaged or mismatched nucleotides in DNA is to create a nick in the phosphodiester backbone of the AP site created when DNA glycosylase removes the damaged base.

46
Q

Xeroderma pigmentosum (XPC)

A

plays an important role in the early steps of global genome NER, especially in damage recognition, open complex formation, and repair protein complex formation.

47
Q

Steps of NER

A
  1. Recognition and binding of the damaged site by a multi-protein complex (two different ways depending on local transcription activity). 2. Local unwinding of the DNA duplex by helicases (parts of the TFIIH protein
    complex) to form a bubble of ~25 bases. 3. Double incision of the damaged strand by two endonucleases and removal of a ~30 base oligonucleotide containing the lesion. 4. Filling in the gap by a DNA polymerase. 5. Rejoining the two ends by a DNA ligase.
48
Q

Transcription factor II Human (TFIIH)

A

is a general transcription factors and unwinds DNA duplex in NER

49
Q

Xeroderma pigmentosum (XP)

A

an autosomal recessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet (UV) light is deficient. One of the most frequent defects in xeroderma pigmentosum is an autosomal recessive genetic defect in which global genome nucleotide excision repair (NER) enzymes are mutated, leading to a reduction in or elimination of NER. characterised by sun hypersensitivity, skin neoplasms, and neurological degeneration (later

50
Q

Cockayne syndrome (CS)

A

characterized by growth failure, impaired development of the nervous system, abnormal sensitivity to sunlight (photosensitivity), and premature aging. Mutations in the ERCC6 and ERCC8 genes are the cause of Cockayne syndrome. The proteins made by these genes are involved in repairing damaged DNA via the transcription-coupled repair mechanism, particularly the DNA in active genes. If either the ERCC6 or the ERCC8 gene is altered, DNA damage is not repaired. As this damage accumulates, it can lead to malfunctioning cells or cell death.

51
Q

Two ways NER machinery recognizes damage

A

Global Genome NER and Transcription-Coupled NER. Steps 1 differs (recognition) while 2-5 are the same. Defects in shared steps (2-5) cause both cancer and CNS disorder.

52
Q

MutS Homologs (hMSH)

A

a tumor suppressor gene and more specifically a caretaker gene that codes for a initiator of DNA mismatch repair (MMR) protein

53
Q

MutL Homologs (hMLH)

A

mediates protein-protein interactions for initiation of mismatch repair (recognition) commonly associated with hereditary nonpolyposis colorectal cancer.strand discrimination, and strand removal.

54
Q

PMS

A

The product of this gene is involved in DNA mismatch repair. The protein forms a heterodimer with MLH1 and this complex interacts with MSH2 bound to mismatched bases. Defects in this gene are associated with hereditary nonpolyposis colorectal cancer. It is involved in mismatch repair and is known to have latent endonuclease activity

55
Q

How does MMR recognize which is the right DNA strand to repair?

A

Newly made DNA is not methylated, but our genome is not as uniformly methylated. Nascent lagging strand is marked by transient 5’ DNA ends of short discontinuous Okazaki fragments. Nascent leading strand is marked by transient presence of ribonucleotides (1 rNMP/1,250 dNMP), which is processed into nicks by RNase H2.

56
Q

RNase H2

A

the major source of ribonuclease H activity in mammalian cells and endonucleolytically cleaves ribonucleotides. It is predicted to remove Okazaki fragment RNA primers during lagging strand DNA synthesis and to excise single ribonucleotides from DNA-DNA duplexes.

57
Q

Lynch Syndrome (HNPCC or hereditary nonpolyposis colorectal cancer )

A

an autosomal dominant genetic condition that has a high risk of colon cancer. The hallmark of HNPCC is defective DNA mismatch repair. HNPCC is one of the most common inherited cancer-susceptibility syndromes and accounts for ~5% of all cases of colon cancers.

58
Q

What is unique about BER?

A

Repairs base damage that does not distort the DNA. Is initiated by damage-specific glycosylases that release the damaged base, followed by removal of the damaged nucleotide.

59
Q

What is unique about NER?

A

Repairs base damage that distort the DNA duplex (e.g. thymidine dimers, carcinogen induced bulky additions to bases). A short oligonucleotides (13-30 nt) including the damaged base are removed via dual incision by two endonucleases.

60
Q

What is unique about MMR?

A

Removes nucleotides that are misincorporated during DNA replication. New strand of DNA is recognized by hemimethylation state in E. coli, and by nicks on newly synthesized DNA strand in humans. Mutations in the MMR machinery cause HNPCC.

61
Q

Lesion Bypass

A

If a cell encounters so much DNA damage of the type that normally blocks DNA replication (such as UV-induced thymidine dimers) that the excision repair systems cannot fix it all, cells resort to a pathway called lesion bypass or translesion synthesis. Lesion bypass allows cells to continue replicating and dividing in the face of immense damage. However, it is highly mutagenic because alternate DNA polymerases that lack 3’ to 5’ proofreading exonuclease activity are used to replicate past the DNA lesion. The result is an error rate 100-10,000 higher than normal DNA replication.

62
Q

Steps of lesion bypass

A

1) The DNA replication machinery stalls behind
a site of base damage. Two “bypass” polymerases bind to the
arrested replication complex. 2) This interaction causes a conformational change
in the replication machinery, placing the bypass
polymerases across from the damaged base.
One bypass polymerase (Pol h) extends synthesis
of the new strand over the lesion. 3) new strand a little further beyond the lesion.
Once the lesion is bypassed, the replication
machinery resumes DNA replication. Mutations are incorporated in the new
strand due to the lack of proofreading activity of
the bypass polymerases.

63
Q

non-homologous end joining (NHEJ)

A

is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, requires no sequence homology between two broken ends, which often leads to insertion or extensive deletion of nucleotides at the breakpoint. Choice between HR and NHEJ varies depending on cell types and cell cycle stages. Recent studies suggest that HR and NHEJ play overlapping roles in DSB repair and cooperate to maintain genome integrity and promote survival.

64
Q

homologous recombination (HR)

A

is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Homologous recombination also produces new combinations of DNA sequences during meiosis. Requires extensive sequence homology between broken ends and template DNA and is generally accurate.

65
Q

PARP

A

The main role is to detect and signal single-strand DNA breaks (SSB) to the enzymatic machinery involved in the SSB repair. Once PARP detects a SSB, it binds to the DNA, and, after a structural change, begins the synthesis of a poly (ADP-ribose) chain (PAR) as a signal for the other DNA-repairing enzymes such as DNA ligase III (LigIII), DNA polymerase beta (polβ), and scaffolding proteins. Amplification of damage signal. Focal enrichment of repair proteins. Change in local chromatin structure.

66
Q

DNA Damage Checkpoint

A

a cellular surveillance mechanism that halts cell cycle progression when DNA is compromised to allow time for DNA repair. It is a signaling pathway composed of damage sensors, signal transducers and effectors. Central to lesion detection is a pair of homologous protein kinases called ATM and ATR, which is recruited to site of DNA damage and initiate the sequential recruitment and activation of downstream proteins. ATM and ATR become activated in human cells undergoing earlier stages of tumorigenesis to delay or prevent cancer. Mutations disrupting this checkpoint result in genomic instability and malignant conversion.

67
Q

checkpoint kinases

A

coordinates the DNA damage response (DDR) and cell cycle checkpoint response. Activation of Chk1 results in the initiation of cell cycle checkpoints, cell cycle arrest, DNA repair and cell death to prevent damaged cells from progressing through the cell cycle.

68
Q

ATM

A

it helps control the rate at which cells grow and divide. This protein also plays an important role in the normal development and activity of several body systems, including the nervous system and the immune system. Additionally, the ATM protein assists cells in recognizing damaged or broken DNA strands. DNA can be damaged by agents such as toxic chemicals or radiation. Breaks in DNA strands also occur naturally when chromosomes exchange genetic material during cell division. The ATM protein coordinates DNA repair by activating enzymes that fix the broken strands. Efficient repair of damaged DNA strands helps maintain the stability of the cell’s genetic information.

69
Q

ATR

A

a serine/threonine-specific protein kinase that is involved in sensing DNA damage and activating the DNA damage checkpoint, leading to cell cycle arrest.[3] ATR is activated in response to persistent single-stranded DNA, which is a common intermediate formed during DNA damage detection and repair. Single-stranded DNA occurs at stalled replication forks and as an intermediate in DNA repair pathways such as nucleotide excision repair and homologous recombination repair.

M2M (23 decks)

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