DNA Replication Organization and Repair Flashcards
(53 cards)
1
Q
Replication and what happens to outcome
A
Copying of DNA and passing on to daughter cells
2
Q
5 theoretical modes of replication
A
1 conservative 2 semi conservative 3 non conservative 4 dispersive 5 end to end
3
Q
Meselson and Stahl experiment
A
-experimented of replication of E.Coli and proved that DNA replicated semi-conservative
4
Q
Enzymes and proteins for DNA replication
A
1 DNA polymerase 1 - repair enzyme which removes errors and growth old gaps in sequence
2 DNA pol 2 - exonuclease function to remove RNA primers
3 DNA pol 3 - adds nucleotides to RNA primer in 5-3 direction
4 Sliding clamp - holds DNA pol 3 in place
5 Ligase - seals nicks between Okazaki fragments
6 Single strand binding proteins - bind single strands at forks and prevents them from winding again
7 Helicase - unwinds double Strand by breaking nitrogenous bases
8 Topoisomerase - prevents supercoiling and relives stress during unwinding
9 RNA polymerase- creates primers to initiate get pol 3 started
5
Q
Replication process summary
A
- origin of replication is identified
- helicase opens up DNA replication creating forks which extend bidirectionally
- Ssb proteins prevent rewinding of coil at forks
- topoisomerase binds region ahead of coil and prevents supercoiling
- primase synthesize primers complimentary to strand
- DNA pol 3 adds nucleotides to primers at 3-OH of primer
- elongating of leading and lagging end occurs
- primers removed by exonuclease action of pol 2
- gaps filled by DNA pol 2
- ligase seals backbone nicks between Okazaki fragments
- reconstitution of chromatin structure including its associated proteins
6
Q
How are origin points identified by origin binding proteins
A
-by a specific sequence of bases
Eg in E.Coli it’s appox 245 base pair region rich in A-T sequences
7
Q
Number of origin points in eukaryotes and prokaryotes
A
- multiple
- just 1
8
Q
Where does energy for the process come form
A
Form nucleotides With 3 phosphates. The phosphate bonds are broken and energy released used to form phosphodister bonds between incoming and growing chains
9
Q
How is origin recognzised by enzymes
A
Specific proteins bind to it
10
Q
Linear and circular DNA in prokaryotes and eukaryotes
A
- chromosomes in eukaryotes is long linear dsDNA bound to nucleoproteins to form chromatin
- prokaryotes have 1 single circular dsDNA supercoiled and has non-histones to compact it into a nucleoid
11
Q
Plasmids and use
A
- extra-chromosomal DNA small and circular in prokaryotes and carriers genetic info
- may or may not be synchronized during replication
-use in DNA recombinant techniques
12
Q
DnaA
A
Binds to site of origin during replication and causes melting ( ATP dependent ) wc results in strand separation
13
Q
What does unwinding by helicase cause and solution
A
- it causes dsDNA to positively supercoil Ahead of fork and negative behind
- the supercoiling affects the unwinding
- solution is topoisomerases which remove supercoiling
14
Q
SSB proteins. Functions, mechanism of binding and properties
A
- they bind to ssDNA and shift the equilibrium in Favor of ssDNA ( not quite an enzyme )
- binding is cooperative ( binding of one molecule makes it easier for another to bind )
-protect ssDNA from nucleases
15
Q
Topoisomerase Type 1, where in gets energy, function, characteristics and use
A
- has strand cutting and sealing activities.
- does not require ATP but store up energy from phosphodister bond when they cleave and use it in sealing
- nick created in one DNA strand and intact passed through before sealing
- relives negative supercoiling both positive and negative in eukaryotes and prokaryotes but only negative in E.Coli E.Coli
16
Q
Topoisomerase Type 2
A
- ATP dependent
- binds to both strands makes breaks and causes stretch of double helix to pass through breaks then seals
- relives both positive and negative supercoiling
17
Q
Gyrase
A
-Topoisomerase with unusual property to introduce negative supercoiling using ATP hydrolysis and counter act positive supercoiling
18
Q
RNA primer
A
- short dsRNA region consisting of base pairs paired to DNA template which initiates action of polymerase 3
- made by primase
19
Q
How does primer initiate synthesis
A
Has free OH group on 3’ end ( acceptor group of a deoxyribonucleotide )
20
Q
What does primase do
A
-converts non-priming proteins into primosome which then makes RNA primer
21
Q
Elongation
A
- pols elongated nascent strand by adding new nucleotides one at a time along ssDNA template
- template specifies sequence at which nucleotides are added
22
Q
Processive and how is clamp formed
A
- polymerase enzyme remain bound to template and do not diffuse away and then rebind again
- due to beta subunit which encircle and forms a ring and moves along template strand forming a sliding clamp
23
Q
Addition of nucleotide substrates and what happens when one type of substrate is depleted
A
- triphosphate nucleotide is taken by polymerase and the enzyme catalyses breaking of tri into mono releasing pyrophosphate.
- reaction releases energy used by polymerase to phosphodister bond between 3-OH of newly synthesized and 5-OH of incoming
-all substrates must present. If one depletes then synthesis stops.
24
Q
Proof reading
A
Polymerase has 5-3 synthesis activity and 3-5 exonuclease activity.
- as each nucleotide is added polymerase ensures it is correct sequence.
- if error made, error removed by exonuclease activity by hydrolytic-ally removing error and add correct one. ( excision )
- must be done is reverse director so 3-5
25
Importance of proof reading
-vital for survival as it avoids mutations
26
Excision of RNA primer
-done by pol 1 through 5-3 exonuclease by hydrolytically removing primer
-pol 1 locates nick between 3 end of new DNA 5 end primer and replaced with dRNA in 5-3 direction.
Also proof reads as it added nucleotides
27
Exo and endonucleases
- removes nucleotides from end of DNA chain
| - from within DNA chain
28
(5-3) and (3-5) exonuclease
- pol 1 can remove 1 or more nucleotides of primer
| - pol 3 can remove only one nucleotide during time of synthesis
29
Termination
- done by specific sequence binding protein tus ( terminal utilization substance )
- tus binds To termination site on DNA stopping movement of pols
30
Diff between eukaryotes and prokaryotic DNA synthesis
- eukaryotes have multiple of origin
| - diff proteins ( but same job )
31
Eukaryotic proteins and their function In DNA synthesis
- origin recognition complex ( ORC ) origin recognition
- minichromosome maintenance ( MCM ) helicase activity
- replication protein A ( RPA ) ssDNA protection
- pol a / primase
- proliferating cell nuclear antigen ( PCNA ) sliding clamp
- RNase H , Flop endonuclease 1 ( FEN 1 ) primer removal
32
Eukaryotic DNA polymerases
Pol alpha - multi subunit of wc one synthesizes primers
Pol epsilon and delta - completes synthesis on leading strand / on Okazaki fragments NB epsilon associates with PCNA
Pol beta and gamma - involved in gap filling / replicates mitochondrial DNA
33
Which eukaryotic polymerase enzymes can’t proof read
Alpha and beta
34
Telomeres, characteristics, properties
- non coding dna and its proteins ( shelterin ) located at ends of chromosomes
- maintain structural integrity of chromosomes
- allow repair mechanisms to recognize true end and breaks in dsDNA
- consists of tandem non coding hexameric sequence -AGGGTT- base paired complimentary C and A’s
- GT region longer
35
Telomere shortening
- following removal of primer from extreme 5 end of lagging end eukaryotes cells can’t replicate then ends of the linear DNA ie no way to fill in remaining gap with DNA and telomeres shorten with successive divisions
- if shorten beyond critical point becomes senescent ( unable to divide )
36
Telomerase
- ribonucleoprotein wc maintains telomeric lengths
| - has protein tect wc is reverse transcriptase and short RNA terc template
37
How telomerase reconstitutes telomeres
- rna base pairs with GT region of ssDNA 3 end and tect ( reverse transcriptase) using RNA template to synthesize DNA in 5-3 direction
- telomerase then translocates to newly synthesized end and process repeats
- once GT region is Lengthened primase pol a uses it to make primer at end of GT region which is then extended in 5-3 direction
- primer removed and nascent strand ligated
38
Reverse transcriptase and retroviruses
- RNA directed DNA polymerase
- involved in retroviruses replication eg HIV
- viruses carry genome as ssRNA and transcriptase uses viral RNA for 5-3 synthesis of viral DNA which then incorporates into host chromosomes
39
Inhibition of protein synthesis by nucleoside analogs
- nucleosides analogs are modified on sugar portion and block chain growth
- by blocking elongation this slows down rapidly growling cells and viruses
Eg AraC ( cytosine arabinoside ) anti cancer chemotherapy
AraA ( adenine arabinoside ) antiviral agent
NB supplied as nucleosides and convert to nucleotides by cellular kinases
40
Types of analogs
- removal of hydroxyl group eg dideoxyinosine ( ddI )
- convert deoxyribose to another sugar ( arabinose )
- substitute sugar on OH with another radical
41
Organization of DNA
- each chromosome 2 m long. DNA and proteins specialize To pack compact it to fit into nucleus
- DNA bound around proteins called histones which pack DNA into functional unit “nucleosome
- Nucleosome’s then arranged into a further complex and condensed into chromosomes that can be segregated
42
DNA packing proteins
- Histones ( H2A H2B H3 H4 ) form octameric core of Individual nucleosome bead
- histone 1 not in core by bound to link dna between beads and facilitates packing of nucleosome into more complex structures
- n terminal are positively charged at physiologic PH due to high contents of lysine and arginine so form electrostatic bonds with -ve 3 and 5 ends
43
Nucleosome and further arrangements
-dsDNA wraps twice around core
Nucleosome packed into nucleofilament ( 40nm fiber ) wc shapes as coil
-fiber arranged into loops anchored on a scaffold and additional organization and condensing leads to chromosome
44
Histone modifications
N terminal of histones is phosphorylated methylated or acetylated
- These reversible modifications influence how tight histones bind to DNA therefore affecting gene expression
- Histone modification is epigenetic’s that is heritable changes in gene expression without changing nucleotide sequence
45
Fate of nucleosome during synthesis
During synthesis nucleosome are disassembled to allow access to DNA for replication and once DNA synthesized nucleosome’s reform rapidly
46
DNA repair And importance
-fixing of errors in genetic code due to error in synthesis or environmental insults to avoid loss of control of proliferating mutated cells ( cancer ) , dysfunctional proteins etc
47
Cause of need for DNA repair
- incorrect base pairs / insertion of extra nucleotides during synthesis and errors escape period reading
- chemical ( delaminates bases ie removes NH3 group ) eg nitrous acids or spontaneous deamination mostly cytosine
- non-ionizing UV ( provides energy to covalently bind adjacent pyrimidines forming dimers )
- high energy ionizing radiation can cause dsDNA breaks
48
DNA repair in summary
- identify lesion ( damage )
- excise lesion
- use sister DNA to synthesize DNA to fill gaps
- ligation
49
Repair of mismatch bases
-Errors in base sequence are not hydrogen bonded
-mut S and L proteins use methylated strand at ( gATC ) nearby sequences to determine parental and new strand.
( daughter strand not methylated immediately so methylated is parental )
-mut H cleaves and cuts daughter strand at error and additional surrounding sequences at 3 and 5 end.
-oglionucleotide removed by exonuclease
-parent strand used as template by pol 1 to fill gap and the ligated.
NB mut and methylated approach for E.Coli
Not certain in people yet
50
Repair due to UV light ( nucleotide excision repair )
- UV specific endonuclease urvABC exinuclease recognizes dimer and cleaves damaged stand at 3 and 5 end
- releasing short oglionucleotide with dimer
- gap filled by polymerase and then ligated
51
UV and cancer
- exposure to unfiltered sun causes dimers in skin which can’t be repaired leading to skin cancers
- can’t be repaired due defects in genes that code NER proteins
- condition called xeroderma pigmentosum ( rare genetic disease )
52
Repairing Base alterations ( base excision repair BER )
1 removal of abnormal base by specific glycosylases hydrolytically from back bone at AP sites ( apurinic or apyrimidnic )
2 specific endonuclease recognize missing base and excise 5 end of AP site ( cut 5 end )
-deoxyribose phosphate lyase removes single base free phosphate sugar and gap filled then ligated
53
Repair of double stranded breaks
1 non-homologous end joining ( NHEJ )
-Group of proteins mediate recognition processing and ligation of the 2 strands of DNA. Some DNA lost in the process so very error prone and mutagenic leading to predisposition of cancer and immunodeficiency syndrome
2 homologous recombination ( HR )
-uses enzymes that normally perform genetic recombination between homologous chromosomes during meiosis much less error prone as any DNA lost using homologous DNA as template
