44 Questions
What are some examples of DNA damage causes?
Ionizing radiation, chemical agents, abortive catalysis
What are the consequences of double-strand DNA breaks?
Inducing genomic alterations
Which enzyme is responsible for single-strand DNA breaks?
Topoisomerase 1
DNA repair mechanisms are specific for different types of DNA damage.
True
Cells repair damaged DNA through _____.
DNA repair pathways
What are XPC and HR23B released to do at other sites with DNA damage?
go to other sites with DNA damage
What happens concomitant with the binding of XPA, RPA, and XPG?
XPC and HR23B are released
The increased ___ distortion produced by XPC/HR23B permits the entry and binding of another transcription factor, TFIIH.
DNA
XPD permits two more proteins to bind during DNA repair.
True
What is the role of AP-endonuclease in the BER pathway?
AP-endonuclease hydrolyses the phosphodiester backbone immediately 5’ to the AP site.
Explain the function of PNKP in the BER pathway.
PNKP phosphorylates the 5’ ends and removes phosphate from the 3’ end.
What is the primary function of Polβ in DNA damage repair?
DNA polymerase β (Polβ) has intrinsic dRP lyase activity.
Which step is included in the NER pathway?
Recognition of damaged site
What are the three objectives of this lecture regarding DNA damage and repair?
- Causes and types of DNA damage, 2. DNA damage repair processes in eukaryotes, 3. DNA damage repair processes in prokaryotes
Which of the following is NOT a type of mutation in DNA?
Intentional mutation
______ is the most often incorporated base across 8-oxoG in DNA.
Adenine
Match the following oxidative stress markers with their significance:
Hydroxyl radical (∙OH) = Marker of oxidative stress Adenine (A) = Most often incorporated across 8-oxoG Guanine = Easily oxidized
Alkylation reagents can lead to lesions that are neutral to the cell.
True
What is the main target of deamination of DNA bases?
Amine-group (-NH2)
Match the following DNA bases with their corresponding replacements:
Guanine = Xanthine Cytidine = Uracil Adenosine = Hypo-xanthine 5-Methylcytosine = Thymine
What is the consequence of an Abasic site (AP site) in DNA?
replication fork stalling, bypassed during translesion synthesis, mutations during semiconservative replication
Which biologically reactive compound leads to Bulky adduct/appendage formation in DNA?
Benzo[a]pyrene
What human genetic disease can be caused by defects in XPA and CSB proteins associated with RNA polymerase?
Cockayne's syndrome
DNA crosslinking can interfere with DNA replication and transcription.
True
Which proteins play special roles in Transcription-Coupled Nucleotide Excision Repair (TC-NER) beyond their roles in Global Genome Nucleotide Excision Repair (GG-NER)?
XPG
XPG and XPF-ERCC1 are specific for junctions between single- and double-stranded DNA.
True
Double-Strand Break Repair can result in two human genetic diseases: _______, and _______.
Xeroderma pigmentosum (XP), Aborted or incomplete DSBR
What is the role of activated CHK2 in DNA damage signaling?
Involved in the activation of p53
Activated p53 induces the expression of the Cyclin-Dependent Kinase Inhibitor (CKI) p21CIP1 gene, leading to inhibition of __________ complexes.
cyclin E/CDK2
Which repair mechanism involves the removal of damaged elements and replacement with new segments of DNA?
Nucleotide Excision Repair (NER)
Base Excision Repair (BER) is the predominant repair mechanism for damaged DNA bases.
True
Match the DNA repair mechanism with its description:
BER = Predominant repair mechanism for damaged bases MMR = Repair of replication errors NER = Removal of bulky adducts and intrastrand cross-links DSBR = Repair of double-strand breaks
What is the major factor that determines the choice of DNA double-strand break repair pathway?
Type of DSB lesion
Which factor affects repair pathway choice at one-ended breaks vs. two-ended breaks?
All of the above
Time is an ________ factor in determining the DNA repair pathway.
important
Poly(ADP-ribose) signalling plays a role in an early response to DNA double-strand breaks.
True
What is the process called where the blunt end of a section of double-stranded DNA is modified by cutting away some nucleotides from the 5' end to produce a 3' single-stranded sequence?
DNA end resection
Resectioning of DNA is performed by MRE11–RAD50–NBS1 (MRN) complex and ______.
Exonuclease 1 (EXO1)
What is the default mechanism for repair by cNHEJ?
Ku70–Ku80 displacement
Synaptic complex formation is part of the cNHEJ repair mechanism.
True
What is the role of DNA end resection in determining the choice between cNHEJ and homologous recombination?
DNA end resection plays a crucial role in determining the choice between cNHEJ and homologous recombination by supporting the displacement of Ku70-Ku80 and engaging long-range resection required for HR.
Which pathways can function opportunistically on ssDNA ends or recombination intermediates?
Both a and b
DNA-PKcs is involved in the cNHEJ pathway.
True
The initiation of nascent-strand synthesis requires an additional ~$______$ minutes.
15
Study Notes
DNA Damage and Repair
- The purpose of this lecture is to introduce students to the causes and types of DNA damage, as well as the processes involved in DNA damage repair in eukaryotes and prokaryotes.
Causes and Types of DNA Damage
- External environmental factors that cause DNA damage include radiation (UV, X-rays, cosmic radiation), chemicals (combustion, water, food), and errors in DNA replication.
- Internal metabolic processes that cause DNA damage include enzymatic reactions, cellular metabolism, and errors in DNA replication.
- Mutations can be classified as neutral, beneficial, or harmful, and can affect cell metabolism and function, leading to disease development or tumor formation.
Types of DNA Damage
- Small-scale mutations include point mutations (silent, nonsense, missense), insertions, deletions, transitions (A↔G, T↔C), and transversions (A↔T, A↔C, T↔G).
- Large-scale chromosomal alterations include amplifications, deletions, and chromosomal rearrangements (translocations, insertions, inversions).
- Oxidative stress is a type of DNA damage caused by reactive oxygen species (ROS), which can lead to mutations and cancer.
- Alkylation of DNA bases involves the addition of alkyl groups (-CH3, -CH2CH3, -CH2CH2CH3, etc.) to the DNA molecule, which can be mutagenic or cytotoxic.
- Deamination of DNA bases involves the removal of the amine group (-NH2) from the DNA base, leading to mutations.
- Loss of DNA bases can occur through spontaneous depurination, DNA damage, or base excision repair, and can lead to mutations.
- Bulky adduct formation involves the addition of biologically reactive compounds to the DNA molecule, leading to mutations and cancer.
- Crosslinking of DNA involves the formation of covalent bonds between DNA bases, interfering with DNA replication and transcription and leading to cell death.### DNA Damage and Repair
Causes of DNA Damage
- Ionizing radiation (X-rays, γ-rays)
- Chemical agents that crosslink DNA
- Abortive catalysis: topoisomerase stalls in the transition state, remains bound to DNA, and cannot relieve supercoiling and reseal the backbone
- Double-strand DNA breaks can induce genomic alterations
Types of DNA Damage
- Single-strand breaks
- Double-strand breaks
- Bulky DNA adducts
- Crosslinks
- Oxidation (8OxoG, Uracil, Single-strand breaks)
DNA Damage Repair in Eukaryotes
- Preserve genome integrity
- Repair mechanisms specific for different types of DNA damage
- DNA damage needs to be detected and signaled to DNA repair machinery
Sensing DNA Damage
- Sensing phase: activation of signaling cascade
- Delay cell cycle progression
- Switch on downstream effectors (specific for the type of damage)
- Cell death via apoptosis
Cell Cycle Checkpoints
- G1/S checkpoint
- G2/M checkpoint
- M phase checkpoint
- Cyclin-dependent kinase inhibitors arrest the cell cycle
DNA Repair Pathways
- Base Excision Repair (BER)
- Nucleotide Excision Repair (NER)
- Mismatch Repair (MMR)
- Double-Strand Break Repair (DSBR)
- Homologous Recombination (HR)
- Non-Homologous End-Joining (NHEJ)
Common Pathway of DNA Repair Mechanisms
- Lesion detection: proteins bound to DNA lesion
- Damaged part removal: nucleases, glycosylases, etc.
- Repair/Resynthesis: DNA ligase, DNA polymerase
- Effects on other cellular processes: replication and/or cell division to allow more time for repair to occur
- Consequence: accurate repair-Survival, inability to repair-Cell death, misrepair-Genome Instability### Direct Reversal Repair (DRR)
- DRR is the simplest step in DNA repair, error-free, and most economical, but limited
- In E. coli, DNA bases are repaired instead of excised via DNA photolyase
- In mammals, DRR mechanisms include:
- O6-methylguanine-DNA methyltransferase (MGMT) which removes mutagenic lesions caused by alkylating agents
- AlkB homologs (ALKBH) family of α-ketoglutarate (α-KG)-Fe(II) dependent dioxygenases
Base Excision Repair (BER)
- BER is the predominant repair mechanism for damaged bases
- Only the damaged base is cut from the target site, unlike NER
- Cleaves N-glycoside linkage
- 5 main enzymes involved in BER:
- N-glycosylases
- AP-endonuclease (APE1)
- PNKP (Polynucleotide Kinase Phosphate)
- DNA polymerase (and flap endonuclease)
- Ligase
Long Patch BER
- Involves displacing synthesis and generating a 5' flap
- PNK phosphorylates 5' end and DNA polymerase β (Polβ) has an intrinsic dRP lyase activity
- PARP (poly ADP-ribose polymerase 1) is involved in long patch BER
Short Patch BER
- Involves non-displacing synthesis
- No 5' flap is generated
Mis-Match Repair (MMR)
- Occurs when incorrect base pairing occurs during replication
- Steps involved: recognition, removal of affected strand, gap repair
- Mutator genes conducting proteins (Mut proteins; MutS /MutL/MutH) play a role in MMR
- MutSα (MSH2-MSH6) recognizes the mismatch
- MutLα (MLH1-PMS2) recognizes the parent strand and complexes with MutSα
- PCNA (proliferating cell nuclear antigen) and RFC (replication factor C) join the assembly
- PMS2 (an exonuclease) makes single-strand breaks near the mismatch
- EXO1 removes the DNA lesion and DNA Polɣ synthesizes the new strand
- Ligase joins the ends
Nucleotide Excision Repair (NER)
- Removes bulky bases, chemical adducts, intrastrand crosslinks, and some oxidative damage
- Steps involved: recognition, binding of a multi-protein complex, local opening of double-helix DNA, dual incision of DNA, gap repair, DNA synthesis, and ligation
- Transcription-coupled NER (TC-NER) occurs in actively transcribed strands and is repaired more rapidly
- Global Genomic NER (GG-NER) occurs elsewhere in the genome
- XPC/HR23B recognizes damaged DNA based on structural distortion
- UV-DDB (UV-damaged-DNA-binding protein) and XPE-containing E3UL are involved in GG-NER
- XPC/HR23B increases structural distortion, permitting entry of TFIIH
- XPB/XPD helicases unwind 20-30 nucleotides including the damaged site
- XPA ensures the site contains damaged DNA
- RPA (single-stranded-DNA-binding protein) protects the separated strands in the open complex
- XPG is a structure-specific nuclease
- Double-strand incision occurs after RPA binding
This quiz covers the causes and types of DNA damage, as well as the processes involved in DNA repair in eukaryotes and prokaryotes.
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