Chapter 13: DNA Replication and Repair Flashcards
What are the four bases in DNA replication?
- Adenine (A) and Guanine (G)= purines
Thymine (T) and Cytosine (C)= pyrimidines
How many hydrogen bonds are present between G and C vs A and T?
- GC = three hydrogen bonds
- AT= two hydrogen bonds
L> less energy to break
What way is DNA read by DNA poly?
- 5’ to 3’
* opposite polarity**
What does the 3’ end of DNA possess?
- free OH and it is important when it comes to adding bases (nucleotides)
DNA stands for?
-deoxyribonucleic acid
Describe DNA
- two long polynucleotide chains
- four chemically similar sub unites (nucleotides/bases)
- hydrogen bonding between bases form double stranded DNA
When is uracil present?
- only in RNA….if it shows up in your DNA it is an indication that there is a mistake going on ….
What can denature DNA?
- helicase and temperature ..making dsDNA nto ssDNA
What makes up a nucleotide?
- 5 carbon sugar
- phosphate group
- nitrogen contaiing base
What are the two nucleotides?
- DNA –> deoxyribose sugar, nucleotides: GACT
- RNA–> ribose sugar, nucleotides: GACU
Nucleoside?
- without a phosphate group
What makes up the backbone of DNA?
- sugar-phosphate
Where will a DNA nucleotide come in?
at 3’ free OH end
Describe Base pairing of DNA.
- Complementary pairing
L> G with C
L> A with T (or U in RNA)
DNA strands run in what type of direction?
- antiparallel bc of polarity
- always read 5’-3’
What two types of grooves are there in DNA?
- Minor groove
- Major groove -
* they are important for the interaction with DNA
* *major groove –>interaction occurs more easily within these groups…molecules can non-covalently bond with the DNA to affect its regulation for replication…protein synthesis
Primary Functions of DNA?
- storage of genetic information
- replication and inheritance
- expression of genetic information
- *genetic info is stored via base pairs
- near identical replication bc of its organization and complimentary base pair interactions
- passed on
- expression of genetic information = proteins
What is a genome?
- complete set of organism’s DNA
- found in every cell
- a genome contains the information to make every protein required by the body
What type of cells do not have a genome?
- red blood cells…. they do not have a nucleus..they are enucleated
How is the genome duplicated in the cell cycle?
- semi-conservative replication
What does IPS stand for?
- induced pluripotent stem cells
What has IPS disproven in terms of our though on genes?
-we use to believe when a cell terminally differentiated they lost all genes not necessary for their functions but this is not the case…they maintain them regardless
Explain the two types of replication DNA was thought to undergo - which was actually proven correct?
- conservative or dispersive replication - disproven. DNA seems to be a mix of original and new DNA that is not identical to the original parental molecule….as replication occurs again and again it becomes less and less identical to the original parental DNA molecule
- semiconservative replication(correct): conserve one of the original parent strands…daughter strand is produced via this…1/2 parent and 1/2 newly synthesized DNA
* *the reason this works is bc of the complementary base pairing..once the parent molecule separates into ss they contain every base needed to be returned back to an identical double helix of the original parent strand
Initiation of Prokaryotic Replication:
-origin of replication in E.coli??
- specific site on the chromosome where replication occurs there …due to high concentration of A and T bonds
- *circular chromosome in prokaryotes
Initiation of Prokaryotic Replication:
- what kind of direction is it?
- bidirectional
Initiation of Prokaryotic Replication:
-What happens at the oriC?
- DNA needs to be denatured and plot into single strands…30 enzymes are involved in this via semi-conservativer explication…replication roles directly through until they pop into two separate chromosomes
Initiation of Prokaryotic Replication:
-replication forks?
L>how many?
- strands separating
- nucleotides are being added
- two forks …opening it up you get two forks going in BOTH directions
DNA Supercoiling:
- what are the states DNA can be found in?
- relaxed DNA
- supercoiled DNA
- negatively supercoiled
- positively supercoiled
- *can cause trouble
DNA Supercoiling:
- Relaxed state?
- what they want to be like..natural state.. proteins etc don’t always let it be this way…but it is the regular helical strand
DNA Supercoiling:
-negatively supercoiled?
- the DNA is under wound..a part of it is actually able to come apart…youd find this sort of at an origin of replication
DNA Supercoiling:
- positively supercoiled?
- essentially what would happen if you ripped the circular DNA apart..whatever is left is all knotted up…extra twisting.. … not good for the DNA
DNA Supercoiling:
- what removes positively supercoils that form during replication ?
- enzymes…
DNA Supercoiling???
he over- or under-winding of a DNA strand, and is an expression of the strain on that strand.
Unwinding and Separation of DNA Strands:
-As DNA unwinds what happens?
- positive supercoiling occurs
Unwinding and Separation of DNA Strands:
- what is DNA gyrase’s function in E.coli?
- as replication continues to expand it is ahead of it reducing tension
- it is a type II topoisomerase
- reversible nuclease aka it is able to cut both strands of DNA..it cuts it (freeing knotted strands) and lets another piece of DNA go through and seals it again aka reversing it
Topoisomerases:
- Topoisomerase I??
- single stranded break
- type 1: cuts through one strand and lets it orate on itself to get out extra coils and then seals it (type 2 deals with knotting)
Topoisomerases:
- how many types?
- two
- type1- cuts one strand
- type 2- cuts both strands
Topoisomerases:
- if there is a mutation in a topoisomerase gene what will happen?
- the organism will not survive it is an essential enzyme (lethal mutation when not properly transcribed and translated)
DNA Polymerases:
-Synthesize what?Direction?
- DNA
- its a replicating enzyme
- 5’ to 3’ direction
DNA Polymerases:
- what does it require to synthesize DNA ?
- template strand
L> it is longer 3’-5’ - primer
L> second strand…needs to be started before polymerase jumps on and connotes synthesizing…initial bases - A and T…going in 5’-3’
DNA Polymerases:
- why is the primer unique?
- it is an RNA strand ….DNA poly needs an RNA primer to jump on bc there is a free OH to extend that second strand
Prokaryotic Polymerases:
-What types are there?
- DNA Poly III
- DNA Poly I
- DNA Poly II
Prokaryotic Polymerases:
- DNA Poly III?
- main enzyme responsible for polymerizing DNA if you were a prokaryote..it was the last to be discovered..bc there is more poly I vs 3…
- extends RNA primers with DNA nucleotides
Prokaryotic Polymerases:
-DNA Poly I?
- involved in DNA repair
- polymerizing activity
- 5’-3’ and 3’-5’ exonuclease activity
- removes wrong bases in DNA when they put the wrong on or if a mutation ….. also when the RNA primer is there it goes in and chops it out and puts on the DNA bases (aka exonuclease activity)
Prokaryotic Polymerases:
- DNA Poly II?
- function is not fully known
- inducing polymerase in SOS response in bacteria
- in the presence of massively DNA damage it will show its head… we can detect it but do not know its function
- thought to help repair massively damaged DNA
Semidiscontinuous Replication:
- Leading vs Lagging strand?
- localized region of DNA
- leading: replicated continuously–> 5’-3’(toward fork)
- lagging: synthesized via back stitching –> 5’ to 3’ (away from fork)
- as synthesis occurs in 5’-3’ away from the fork there are gaps appearing as the DNA is opened up more with the poly synthesizing in another area
DNA templates and nontemlpates:
-hair pin loops?
- when a strand folds back on itself bc it was complimentary…
Okazaki Fragments??
- these are found only on the lagging strand
- 1000nt segments are synthesized at a time
- fork opens up..you have a bare section of DNA so another round of synthesis needs to occur…replication fork will open up 1000nt and then it comes in and fills it up
What Proteins are involved with the Replication Fork? (6)
- DNA Polymerase
- DNA Helicase
- Single Stranded DNA-binding proteins
- DNA Primase
- DNA ligase
- DNA sliding clamp
DNA Helicase??
- DNA unwinding enzyme
DNA Helicase:
- describe Dna B and C helicase
- 6 subunits that encircle ssDNA
- attaches to ssDNA at origin of replication via Dna C
- Moves in a 5’-3’ direction as png as there is a replication fork on the lagging strand. aka it attaches to the lagging strand
SS-DNA Binding Proteins??
L> what are they?
- Helix destabilizing proteins
- affinity for ss-DNA
- prevent hairpin loop formation, rewinding or damage to strand
AKA Helicase denatures/destabilizes DNA but these stabilize it
Do ssDNA binding proteins prevent enzymes from getting into the DNA?
no
DNA Primase??
- this enzyme synthesizes short RNA primers in empty/ DNA free spaces on the laying strand
- approximately at every 1000 nucleotide stretch on the lagging strand
- provides 3’ OH end for DNA polymerase III ….forms part of the primosome which also includes helicase
Why is it important that DNA primase puts down RNA and not DNA?
- just incase there is a mutation…initial synthesis makes a ton of mistakes…ideally it just wants free OH doesn’t care how it gets to it…this also doesn’t slow down synthesis
DNA Ligase?
- enzyme that seals “nicks” in the DNA strands sugar phosphate backbone
- *it is an RNAse H repair enzyme
When does ligase come in?
- after an RNA primer has been fro moved from the okazaki fragment by DNA Poly I
Sliding clamp?
- beta clamp attaches DNA poly to ssDNA section
- it assembles on the RNA primer-DNA template junction
- It is a multisubunit clamp loader
- *RNA DNA hybrid section
Why is there a sliding clamp?
- to ensure that the polymerizing enzyme does not fall off and it ensures the ply stays where it is suppose to be until it come in contact with the primer of the next O fragment..as soon as its there …there is a stall and it dissembles
DNA Poly III Holoenzyme ?
- the replication machine
DNA Poly III Holoenzyme
-what makes up the replisome??
- DNA Poly III
- t subunits
- beta clamp
- clamp loader
- *DOES NOT INCLUDE HELICASE
- ensures everything is together as it slides along the replication fork
Eukaryotic Replication:
- replicons?
- a DNA molecule or RNA molecule, or a region of DNA or RNA, that replicates from a single origin of replication
Eukaryotic Replication:
- foci?
Replication foci are subnuclear sites where DNA replication takes place.
Eukaryotic Replication:
-Replicons
L>what affects mammalian replication?
- gene activity (active genes replicated first in S phase
- chromosomal level of compaction (heterochromatin -replicated later in synthesis phase )
Eukaryotic Replication:
- make a comparison with prokaryotes with respect to oriC
- in prokaryotes there is only one origin of replication in eukaryotes there are many
Eukaryotic Replication:
-Replicons
L> why are active genes replicated first?
- bc they are more accessible bc they are not as packed away bc they are already being used in the cell
- genes that are not being used will be done later (heterochromatin(tight packed regions of chromosomes))
- **this shows that the sequence of DNA does not dictate what will be replicated first or last
Eukaryotic Replication:
-Replicons
L> XX chromosomes?
- one in every cell will be compacted away so it is replicated later …so the sequence in the active chromosome will be replicated first …they both have the same sequence but the level at which it is packed away determines how early/late it will be replicated in S phase..
Initiation of Eukaryotic Replication:
-Origin Recognition Complex?
- ORC
- bound to an autonomous replication sequence (ARS)
L>molecular landing pad - aka protein binds to a particular sequence (ARS)..on its own it will start synthesis
**found this out by splicing it out of yeast and putting it in bacteria - anywhere it was put replication occurred.
Initiation of Eukaryotic Replication:
-Pre-replication complex?
- licensing factors
- Mcm proteins (2-7)
- *mcm proteins = licensing factors ( 6 of them) mini chromosome maintenance proteins bbd to the ORC and ARS
Initiation of Eukaryotic Replication:
- Activation of protein kinases?
-Cdk (cyclin dependent kinases) levels remain high to keep up ignition
Initiation of Eukaryotic Replication:
- are mcm proteins able to re-associate with the replicated ORC?
- NOPE
(2N-4N…8N)
**they are exported so re-replication does not occur
Initiation of Eukaryotic Replication:
-steps?
- ORC bound to ARS
- Pre-replication complex
- Activation of Protein kinases
* *after these = replication fork occurrence
* **Replicative helicase?
Initiation of Eukaryotic Replication:
-In vertebrates initiation of replication is regulated by?
- ORC removal
- high Cdk levels
- export of Cdc6
- inactivation of Cdt1
- *not sure of mcm proteins are exported in verts
Eukaryotic DNA Polymerases?
- alpha - associates with primes aka initiates synthesis of okazaki frags
- beta - functions in DNA repair
- gamma - replicates mitochondrial DNA*
- delta- primary DNA synthesizing enzyme* -> on lagging strand
- epsilon- nuclear DNA replication *–> on leading strand
- there is 3’-5’ exonuclease proofreading ability
- ***without epsilon replication would not occur
Eukaryotic Replication Fork:
- PCNA?
- proliferating cell nuclear antigen (sliding clamp)
Eukaryotic Replication Fork:
- RFC?
- replication factor C (sliding clap loader)
Eukaryotic Replication Fork:
- RPA?
- replicating protein A (ssDNA binding proteins)
Eukaryotic Replication Fork:
-FEN-1?
- endonucease that cuts RNA primer ‘flap’ displaced by poly delta
L> inside a strand of DNA it can just jump into a pile of nucleotides and cut it doesn’t need an end
*when delta jumps on to lagging strand it will jump on to the primer which is actually DNA and RNA and will finish off O fragment…poly delta will plough through the RNA nucleotides creating a flap..and clean it up via FEN-1
List and explain the functions of the proteins required for replication in eukaryotes!
- ORC proteins: recognition of origin of replication
- Topoisomerase I/II - relieves positive supercoils ahead of replication fork
- Mcm - DNA helices that unwinds parental duplex
- Cdc6, Cdt1 - loads helices onto DNA
- RPA - maintains DNA in single stranded state
- RFC - sub units of the DNA poly holoenzyme that load the clamp onto the DNA
- pol delta/epsilon- primary replicating enzymes; synthesize entire leading strand and Okazaki frags; have proofreading capability
- PCNA - ring shaped subunit of DNA poly holoenzyme that clamps replicating poly to DNAl works with pol III in E.coli and pol deal or epsilon in eukaryotes
- Primase: synthesizes RNA primers
- poly alpha : synthesizes short DNA oligonucleotides as part of RNA-DNA primer
- DNA ligase: seals okazaki fragments into continuous strand
- FEN- 1 removes RNA primers; pol I of E.coli also fills gap with DNA
What are the proteins that are involved with prokaryote replication and parallel with eukaryotic ones?
- DnaA (ORC)
- Gyrase (Topoisomerase I/II)
- DnaB(Mcm)
- DnaC(Cdc6,Cdt1)
- SSB(RPA)
- y-complex(RFC)
- Poly III core( Poly delta and epsilon)
- Beta clamp ( PCNA)
- DNA ligase (DNA ligase)
- Poly I (FEN-1)
* *brackets = eukaryotic parallel
* *these have the same functions as listed in card asking about proteins /functions in eukaryotic replication
* *poly alpha is unique to eukaryotes
Nucleotide Excision Repair:
- functions?
- damage recognition
- strand separation
- incision
- excision
- DNA repair synthesis
- Ligation
Nucleotide Excision Repair:
- are these repair systems perfect?
- nope
- overtime things can go wrong…x-rays ionization radiation, absorption of thermal energy from metabolism can split A and T from the backbone
- UV: T dimers
- C and G can be switched around
- radiation from non-ionization radio waves ..microwaves…visible light (UV is more dangerous) X-rays and gamma go into deep tissue
Nucleotide Excision Repair:
- how many bases in a thousand escape change?
1
Nucleotide Excision Repair:
- how does removal of “bulky legions” occur?
- Two pathways
1. Transcription-coupled pathway
2. Global pathway
Nucleotide Excision Repair:
-removal of “bulky legions”
L> Explain Transcription-coupled pathway
- the cell that is transcribing at the time is transcribing the genes it needs to be expressed so as RNA poly it can stall if there is a lesion this signals that we need to fix the problem bc that is actually a gene that is required for the cell…quick and easy way to get in and fix it right away
- *Any type o lesions can be signalled by a stalled polymerase ensuring the genes of greatest importance in that cell are fixed first
Nucleotide Excision Repair:
-removal of “bulky legions”
L> what is a lesion?
- region of DNA that is wrong
Nucleotide Excision Repair:
-removal of “bulky legions”
L> Explain the Global pathway
- scans DNA all over - no one particular section is more important…it is slower and less efficient than the other pathway ut it will correct the DNA strands in the rest of the genome for that cell
Nucleotide Excision Repair:
-removal of “bulky legions”
L> CSB?
L> XPC?
- chromatin remodelling protein
- gene product involved in DNA repair
Nucleotide Excision Repair:
-removal of “bulky legions”
L> how does it occur for both coupled and global pathway?
- TFIIF - transcription foactor for separatinn
- strand is separated via TFIIF and XPB and XPD (helicase)
3. XPG on 3’ end - XPF-ERCC1 on 5’ end
- incision= cutting of lesion
- excision = removal
Base Excision Repair:
-DNA glycosylase?
- recognizes inappropriate base
- base removed via cleabvage go glycosidic bond
Base Excision Repair:
- AP endonuclease?
- cleaves the backbone of DNA strand
Base Excision Repair:
-Beta Polymerase
- removes sugar phosphate and fills gap
- empty spot and a 3’OH and adds in correct base that is complimentary
Base Excision Repair:
-DNA ligase?
- seals the strand
Base Excision Repair:
- steps?
- DNA glycosylase
- AP endonuclease
- Beta polymerase
- DNA ligase
Xeroderma pigmentosum?
- inability to repair DNA lesions caused by UV radiation
- 7 enzymes that could have a potential mutation
- problem with DNA repair systems
