DNA: Introduction, Structure, Replication, & Repair (Gelinas) Flashcards
(89 cards)
1
Q
3’-5’ phosphodiester bond between:
A
3’-OH sugar of one nucleotide to 5’-P on other. One
5’ end: free phosphate;3’ end: OH
2
Q
___bp/turn
Complete turn every ___Angstroms
A
10 bp/turn
Complete turn ever 34 A
3
Q
Major and minor grooves ____
His tones are in the ___ grooves
A
- major and minor grooves help regulate gene expression because proteins bind here
- histones are in the minor grooves
***certian anti cancer drugs exert cytotoxic effects by intercalating into minor groove of DNA & interfere w DNA and RNA synthesis`
4
Q
DNA topoisomerases
A
- change tertiary structure of DNA
- introduce swivel points: transiently break one or both DNA strands, pass strands thru break & rejoin them
- can remove positive and negative super oils
5
Q
DNA TopoI
A
Cut single strand, NO ATP
Can remove +/- supercoils
6
Q
DNA Topo II
A
- cuts both strands, needs ATP
- can remove +/- supercoils
7
Q
bacterial DNA gyrase
A
- unusual Topo II
- can introduce - supercoils as well as removing +/- supercoils
- needs ATP
8
Q
What blocks DNA gyrase
A
Quinolone so; selectively inhibit bacterial DNA synthesis , have fewer side effects bc we don’t have it
**chemotherapetuics convert topoisomerases into DNA breaking agents –> DNA breaks & cell death
9
Q
Histones in nucleosome
A
H2A, H2B, H3, H4
-basic( Arg & Lys RICH)
10
Q
H1
A
DNA spacer
11
Q
Euchromatin/heterochromatin compaction is affected by
A
Histone modification (acetylation/methylation)
Ex: as soon as DNA replication is done; H1 binds spacer DNA & promotes tight packing of nucleosomes –>winds into solenoid (nucleofilament)
Solenoid loops onto itself –> large DNA loops & protein scaffold –>causes the 4 arm structure of classic metaphase chromosome
12
Q
Prokaryotic DNA
DNAP I
What does it do and exonuclease activity?
A
Replication (primer removal & gap synthesis) + Repair
Exonuclease activity: 3’ to 5’ and 5’ to 3’ (only one that is also 5’ to 3’)
13
Q
Prokaryotic DNAP II
What does it do & exonuclease activity?
A
Repair
3’ to 5’
14
Q
Prokaryotic DNAP III
What does it do and exonuclease activity?
A
Replication (leading & lagging strand)
3’ to 5’
15
Q
Prokaryotic DNA Replication Initiation
A
- Initiation: opening @ rich A-T origin, recognized by DnaA which melts this in ATP-dependent manner
* E.Coli replicated from a single origin
16
Q
Prokaryotic DNA replication: DNA Strand separation is _______ with ____replication forks at origin
Catalyze do by ____ in pre priming complex
A
DNA strand separation is bidirectional with 2 replication forks at origin
Catalyst by DNA helicase (DnaB, which binds near replication forks and uses ATP to force strands apart)
SSBs bind cooperatively to keep strands apart & protect from uncleared
17
Q
______ (ex: DNA gyrase) works ahead to relieve tension by removing + super oils
A
DNA TopoII
18
Q
_____ are needed to initiate DNA synthesis
A
RNA primers
- RNA primase synthesizes short RNA primers (5’ to 3’)
- ->provide free 3’-OH as acceptor of 1st deoxyribonucleotide
-primers continuously synthesized at replication fork on lagging strand
19
Q
____ recognizes RNA primer
A
DNAP III
20
Q
DNA synthesized in ____ direction
A
5’ to 3’
Short Okazaki fragments are 5’ to 3’ away from fork
21
Q
Elongation catalyze do by _____
A
DNAP III
0until blocked by RNA primer
22
Q
How is a phosphodiester bond formed
A
Nucleophillic attack of 3’-OH on a 5’ Phos with formation of PPi group
-DNAP I makes bond on 3’-OH; DNAP III on 5’-P
23
Q
Proofreading after DNA replication by
A
3’ to 5’ exonuclease activity of DNAP III to remove erroneous nucleotides
-after DNA rep, MMR can replace mismatched nucleotides
24
Q
RNA primer excision & ligation by
A
5’ to 3’ of DNAP I removes primers from Okazaki fragments & can then proofread ( has 3’ to 5’ & 5’ to 3’) exonuclease activity
DNA ligament joins Okazaki fragments, ATP dependent
25
Origin of replication for eukaryotic DNA
Multiple origins
| -2 replication forks at each origin
26
DNAP alpha
Exonuclease activity?
- eukaryotic primer synthesis
- contains primase, synthesis on leading and lagging
- no exonuclease activity
27
DNAP delta
Exonuclease activity?
Eukaryotic DNA replication on lagging
-associates with PCNA *processivity factor for proliferating cell nuclear antigens* to elongate lagging strand
- displace 5' primer of Okazaki fragment (later this is degraded by FEN1 (flap exonuclease))
- 3' to 5' exonuclease activity
28
DNAP epsilon
- eukaryotic DNA replication on leading strand
- associated with PCNA to elongate leading strand
- 3' to 5' exonuclease activity
****if dysfunctional, DNAP epsilon can substitute
29
Which eukaryotic DNAPs also involved in DNA repair?
DNAP delta & DNAP epsilon involved in MMR, NER
30
Do histones remain associated with parental strand as replication fork advances?
Yes, and new ones are synthesized simultaneously with replication
31
Telomeres are made up of no coding ____DNA repeats
G-rich (TTAGG)
32
Roles of telomeres
- protect ends of linear chromosomes from:
- degradation, recombination, end to end fusion, prevent loss of coding DNA during replications (DNA lost when RNA primers removed(
33
How are telomeres added?
Telomerase adds G-rich DNA repeats (TTAGGG) to single stranded 3'-ends
34
Telomerase has ____ activity
Reverse transcriptase; can synthesize DNA from RNA template
-terminal extension allows extra room for primer to bind later on & initiate lagging strand synthesis on other strand
35
Telomerase is implicated in cell aging, cancer, how?
- active in cells pre-birth, stem cells, germ cells post birth
- inactive in most somatic cells, telomeres shorten with each division
- reach critical point, can have chromosome end-to-end fusion, and p53 can induce cell growth arrest to prevent this from happening & reslt in genomic instability
36
What happens if telomerase reactivated and there is a loss of p53?
Cancer; there is uncontrolled growth
-telomerase inhibitors can limit human cancer proliferation
37
Dyskeratosis Congeinta
Reduced telomerase activity
-affects precursor cells in highly proliferative tissues (telomerase maintains telomeres, esp in rapidly dividing tissues)
38
Symptoms of patients with dyskeratosis congenita
Patients generally die from bone marrow failure due to loss of hematopoietic renewal
-highly proliferative tissues are especially affected (hair, oral, gut, lung, hypogonadism, rail to produce RBCs)
39
Hutchinson-Gilford Progeria
- inherited
| - accelerated telomere shortening
40
Hutchinson-Gilford Progeria symptoms
- alopecia, aged skin, short, accelerated atherosclerosis
| - patients generally die from MI before age 20
41
Base Excision Repair repairs...
single base
Base modifications:
1) deamination (C-->U)
2) depurination (releases guanine/adenine from DNA)
Alkylation, oxidation, ROS
42
BER mechanism
DNA glycosylase initiates this: AP endnonuclease cuts PD backbone, base removed, DNAP fills in new DNA & seals knick
43
Defects in BER
- mutation in gene encoding DNA glycosylase (MYH) --> high risk for colon cancer (adenovirus colorectal polyposis syndrome)
- mutation in RecQ family (?)
44
Single strand break repair
- specialized pathway of BER
| - usually accompanied by loss of single nucleo tied & damaged 5' or 3' termini at site of break
45
Source of most SSBs
ROS
46
PARP1 and XRCC1 in SSBR
PARP1: detects disintegration of oxidized deoxyribose and binds and is activated
XRCC1: molecular scaffold for multiple repair proteins -->stimulates enzyme components & accelerates process
47
Why do SSBs need to be repaired rapidly?
Can result in:
- collapse of DNA replication fork during S phase --> DSBS
- stalled transcription
- increased cell death thru PARP1 activators
48
Ataxia coulomb tour apraxia (AOA1)
1) molecular defect
2) DNA Repair pathway affected
3) characteristic features
1) aprataxin (APTX) (can't fix 5' breaks)
2) single stranded break (SSB) repair (APTX processes 5' end breaks)
3) variable onset (1-16 yrs), cerebellum atrophy, ataxia, oculomotor apraxia, late atonal peripheral neuropathy
- autosomal recessive
- lacks non-neurological features (unlike A. Telangiectasia)
49
How does Ataxia Oculomotor Apraxia differ from Ataxia Telangiecstasia?
- no immunological deficiency unlike AT
- AT has more instances of cancer than AOA1
- AT hypersensitive to XRAYS
- AT more cancer (AOA1 has redundancy, other ways to repair 5' ends, so less genomic instability)
50
Why are mutations in SSBR largely restricted to nervous system?
- neurons more dependent on APTX for DNA end-processing
- high levels of oxidative stress encountered by nervous system, low levels of anti-oxidant enzymes
- high transcriptional demand in post-mitocic neurons, further dependency on SSBR machinery
51
What is repaired in NER?
Bulky adducts, dimers, photo products, chemical adducts
**repairs damage by any large change in structure of DNA double helix
52
NER method
1) multi enzyme complex scans DNA for distortion in double helix
2) PD bond on both sides cleaved by excinucleases(UVR/XP)
3) oligonucleoitide containing lesion removed by DNA helicase
4) gap repaired by DNAP & ligament
53
TC-NER repair proteins
CSA, CSB; recognizes stalled RNAP II, ubiquitinate the RNAP II for destruction
54
Defects in TC-NER primarily affects
CNS
---> developmental and neurological disorders
55
GG-NER pathway proteins
Recognition by XPC, XPE repair proteins
-recognize helix distortions caused by damage
-repair by endnonuclease, helicase XPD removes Oligomer, DNAPI/delta/epsilon, ligase seals
56
Main symptom in GG-NER path mutations
Skin cancer
| -DNA replication arrest leads to genomically unstable cells
57
Common path of TC-NER and GG-NER at which proteins
XPA, RPA
Leads to cancer and CNS disorder
58
Xeroderma pigmentosum mutation
GG-NER proteins (XPE, XPC-->cancer)
**these are the proteins that detec helical distortions)
Or
GG-NER/TC-NER common pathway proteins (XPA, XPD --> cancer and CNS)
59
Symptoms of xeroderma pigmentosum
- extreme solar sensitivity
- 2000x risk of skin cancer
- autosomal recessive
- symptoms start at 1-2 years
- 10 to 20x inc in internal neoplasms (brain/lung/gastric tumors)
**pts with common pathway have neurodegenerative symptoms
60
Proteins unique to GG-NER
XPC, XPE --> cancer
61
Proteins unique role to TC-NER
CSA, CSB
62
Common elements in TC-NER + GG-NER
XPA, XPB, XPD, XPF, XPG --> both CNS disorder & cancer
63
Cockatiel syndrome mutation
CSA, CSB proteins --> these are proteins that recognize DNA damage in transcription ally active regions
-helix distortions blocks RNAPII and the stall helps to initiate TC-NER repair
- transcription does not recover when blocked -->severe developmental and neurological symptoms
- RNAP arrest inducer of apoptosis
-no cancer even though photosensitive
64
Mismatch repair corrects mutations in
Replication
G:C--> G:T (mismatch)
G:C--> G:CC (extra)
*no damage!
65
Repair proteins in mismatch repair
MSH2/6 (mismatch)
MSH2/3 (insertion/deletions)
(these are called Mut in prokaryotes)
-these recognize error
Endocluease (MLH1/PMS2) cleaves and helicase/exonuclease remove
-DNAP III (delta/epsilon + PCNA) fills gap and ligase seals
In eukaryotes, new strand contains nicks
In prokaryotes, new DNA error not immediately methylated (non-methylated piece gets cut out)
66
90% of LYnch patients have a mutation in
MSH2 or MLH1
- patients prone to colorectal cancer and other cancers
- defects in MMR --> cancer
67
MMR deficient cells have instability at ____ regions
Micro satellite regions (MSI)
68
MSIs
-repetitive DNA sequences 1-4bp that are particularly susceptible to DNA replication errors when MMR absent
69
MSI instability results in
Production of new alleles from unprepared replication errors
70
MSI is a diagnostic marker for
- loss of MMR activity in tumor cells--> PCR analysis and compare length
- unstable if distribution of fragments of tumors differ from normal tissue
71
Hall mark of Lynch syndrome tumor cells
MSI
72
Test for Lynch syndrome
Amsterdam criteria
| Molecular genetic testing for Germaine mutations in MMR genes
73
Characteristics of lynch syndrome
```
Autosomal dominant
Early onset (
```
74
DSBS induced by
Stress (ionizing radiation, oxidizing agents ,replication errors, metabolic products
-antineoplastic drugs (bleomycin, anthracyclin, topoisomerase inhibitors)
75
How to visualize DSBS
Immnochemical staining for foci enriched in phosphorylated histones (gamma-H2AX)
76
DSBS can be repaired by _____ or ______
HHomologous recombination (HR)or non-homologous end joining (NHEJ)
77
Homologous recombination requirements
-use info on sister chromatid or homologous chromosome to align DNA
78
In mammalian cells, HR is restricted to
S and G2 phases (when sister chromatid present
79
Mechanism of DSB-HR
Requires several recombination proteins
- recognizes areas of sequence matching btwn 2 chromosomes, brings them together
- DNA replication process uses undamaged chromosomes as template to transfer into to broken chromosomes
80
Hallmarks of NHEJ pathway
-major pathway
-doesn't require homo chromosomes
-error prone
-rejoins what remains of 2 DNA ends; since it tolerates nucleotide addition/loss at rejoining site, it is imprecise --> leads to accumulation of random mutations
"Information scars"
-PD bond structural integrity is restored
81
What recognizes breaks and recruit DNA PKcs?
In NHEJ, Ku70/Ku80 binds DNA ends and facilitate recruitment of DNA PKCs
- WRN Helicase facilitates strand opening
- frayed ends removed by DNA-PKCs
-DNA Pol u and lambda repair ( DNA Ligase IV and XRCC4 glues it together)
82
Mechanism of HR repair
RAD52 binds DNA ends, Rad51 recombinanse searches for homologous and aligns
Nucleohelicases make knick (RPA, RAD52, BRAC2) and RAD51 promote single strand invasion into homo DNA nucleases
83
What regulates RAD51 recombinase
BRCA11, BRCA2
84
Mutation in Ataxia Telangiectasia
ATM Kinase (facilitates entry of DNA repair machinery into heterochromatin)
*DSBR affected
When mutated, persistent DSBs localized to heterochromatin
Normally, ATM kinase signals to cell cycle checkpoint to slow passage of cells thru cycle and responds to physiologic breaks during development/differentiation of B and T cells (VDJ recombination)
When mutated ---> inc chromosomal abnormality items in B and T cells, why 30% of AT pts. Develop lymphoid tumors
85
Symptoms of ataxia Telangiectasia
- autosomal recessive
- neurological impairment
- cerebellar ataxia
- variable immunodeficiency
- lymphoid tumors
- X-RAY hypersensitivity -->Cancer
- ocular and cutaneous Telangiectasia (small dilated blood vessels near surface of skin/mucous membranes usually around face)
86
RecQ helicases (WRN)
- essential roles in DNA repair
- may interact with specific DNA repair pathways
- processes telomeres DNA and activates DNA damage responses (recombination, repair, replication) to prevent DNA damage
87
Mutation in Werner Syndrome
WRN Helicase (RecQ helicase) (involved in BER, DSBR
88
Symptoms of Werner's Syndrome
Autosomal recessive
Early onset of aged appearance + age related disorders
Clinical sign: lack of pubertal growth (ex: enter high school normal height, everyone grows except they)
- sarcomas, hypogonadism, health declines death :47-54
- telomere shortening, chromosomal rearrangements -->susceptible to malignant transformations and frequent telomere fusions
89
SSB Repair Pathway
1) recognition by PARP-1
2) recruitment of XRCC1 (scaffold for multiple repair proteins)
3) many enzymes (APTX) process 3'/5' ends
4) restoration of proper 3'-OH and 5'P groups
5) DNAPolB & ligation
