DNA Replication and Repair Flashcards by Adam Purviance

Replication of DNA occurs in a ____-_____ manner

semi-conservative

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______ replication of DNA is a model where each DNA strand serves as a template for DNA synthesis, but the original DNA remains intact

Conservative

note: if this model was correct, there would be no DNA molecules composed of parental an daughter strands together in one molecule

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Each new DNA molecule consists of an old strand (the template) and a new strand in semi-conservative replication. True or false?

true

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Bacteria have single chromosomes which are circular and possess a unique replication origin. True or false?

true

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Eukaryotic chromosomes have how many separate origins of replication per chromosome?

1000-2000

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Bidirectional replication (two replication forks proceeding in opposite directions) does not occur in DNA. True or false?

false; it does

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DNA replication proceeds only in the ___ –> ____ direction

5’ –> 3’

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DNA replication is initiated from pre-existing ____ (short section of RNA which is complementary to the template strand)

primers

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DNA synthesis proceeds from discreet sites meaning it must unravel two parental DNA strands at particular sites and use each single strand as a ___ to direct synthesis of new complementary daughter strands

template

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In this strand, replication is continuous

leading strand

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in this strand, replication proceeds from multiple primers and results in forming short DNA sequences which are eventually joined; synthesis is discontinuous

lagging strand

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____ problems must be overcome in DNA replication. Both prokaryotic and eukaryotic DNAs exist not as linear molecules with free ends but rather as closed-loop molecules. ____ of the DNA molecule during replication creates torsional strain which must be removed for replication to proceed

topological; supercoiling

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In regards to prokaryotic DNA replication, DNA polymerases catalyze the following reaction:

(dNMP)n + dNTP —-> (dNMP)n+1 + PPi –> 2 Pi

Only _______ ____ can serve as substrates

deoxyribonucleic triphosphates

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All DNA polymerases require both a template and a primer. True or false?

true

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During DNA replication in both prokaryotic and eukaryotic cells, both of the strands of the parent DNA molecule serve as _____ and small fragments of RNA serve as ____

template; primer

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In regards to prokaryotic replication, RNApolymerase (primase) is necessary for what?

the synthesis o the RNA primers required for DNA replication

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In regards to prokaryotic replication, DNA polymerases require a free ___-___ terminal from which to start. The RNA polymerase adds an oligonucleotide from 10-60 bases to serve as a ____.

3’-OH; primer

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The short sections of primer RNA plus DNA which form on the lagging strand are called what?

okazaki fragments

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During DNA replication, unwinding of DNA must occur as well as strand separation. The enzymes which carry out unwinding are known as _____

helicases

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The helices bind to sing-stranded DNA and require ____ which is hydrolyzed in order to drive enzyme function

ATP

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Define replisome

total proteins and enzymes required for DNA synthesis at replication fork

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During DNA replication, unwinding of DNA creates ___ supercoiling which must be removed by enzymes called ____

positive; topoisomerases

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Topoisomerases catalyze interconversion of different topological isomers of DNA. These enzymes relieve ____ ahead of the replication fork

tension

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Explain the difference between Type I and Type II topoisomerases

type I: makes a break in one strand of the DNA helix and passes the other strand though the break to relax the supercoil. The break is then sealed. Doesn’t require ATP

type II: also known as DNA gyrase - produces an enzyme-bridged break in both strands of DNA. Another region of duplex DNA is passes through the gap by the enzyme, thus two supercoils are removed in one step. This enzyme requires ATP

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Topoisomerase ___ is the target molecule for some of the most important anti-cancer drugs

Ciproflaxacin is a widely used antibiotic which is active against ____

gyrase

____ proteins keep the separated strands as single strands. As DNA replication proceeds, these proteins are displaced and reused. The affinity for these proteins for single stranded DNA is 1000 X greater than double-stranded DNA.

SSB

This enzyme is responsible for extending the growing DNA strand until the 5' ribonucleotide of the primer of the previously synthesized precursor fragments is reached.

DNA polymerase III

Once the precursor fragment is reached, DNA polymerase III can go no further. This enzyme takes over from here and removes RNA primer as it lays down deoxyribonucleotides in the same place

DNA polymerase I

DNA polymerase III is processive. What does this mean?

Once DNA polymerase III is bound to the template, it probably never dissociates until the entire chromosome has been replicated

How many DNA polymerase molecules function at each replication fork?

2 | note: 4 in replication bubble with both forks

DNA polymerase III is active as a holoenzyme composed of ___ subunits

DNA polymerase I has three enzymatic activities. What are they?

1) possesses a 5'-3' exonuclease activity that cuts out the RNA primer 2) Fills in cut out primer with the dNTP matching the exposed DNA template 3) possesses a 3'-5' exonuclease activity for proofreading

What is the replication error rate for prokaryotic replication?

wrong base 1 in 10,000 elongation steps (10^-4 error rate)

What is the error rate of the exonuclease activity of DNA polymerase I in prokaryotic replication?

10^-3

The combined error rate for prokaryotic replication is ___

10^-7; once in every 10 million bases

This enzyme seals the nick between the fragments of newly synthesized DNA

ligase | note: catalyzes the synthesis of a phosphodiester bond between a 3' hydroxyl group and a 5' phosphate group

True or false? DNA ligase can bridge a gap, i.e.: fill in a missing nucleotide or a base

false; it can't do that

What 3 events occur to initiate DNA replication at oriC in E. Coli?

1) DNA A proteins bind to oriC to initiate replication by separating both strands 2) DNA B + DNA C complex (DNA B is a helicase) bind to the separated strands to form replisome 3) primase joins the complex to begin synthesis of RNA primer

In regards to Eukaryotic DNA replication, multiple DNA polymerases have been identified in eukaryotic cells. These are DNA polymerase alpha, beta, gamma, and g/epsilon. Give the location and function for each

alpha: nucleus; responsible for formation and extension of RNA primers beta: nucleus; involved in DNA repair in the nucleus gamma: mitochondria; replication of mitochondrial DNA

Polymerase ____ synthesizes DNA on the leading strand

delta

The association of the nucleosome and DNA at the site of DNA synthesis is weakened through ____ and ___ of the histones (makes them more negative)

acetylation; phosphorylation

E. Coli replicates at about _____ bp per minute and eukaryotes at about ____ to ___ bp per minute

100,000; 500-5,000

The inequity of eukaryotes in replication rate is overcome through having many ___ sites (perhaps 2000) per chromosome rather than one

initiation

To activate an origin, ____ must bind at the origin of replication

ORC (origin replication complex)

Helicase and SSB prepare the origin region for insertion of DNA ____-____. A ____ factor must also be bound near each origin to ensure the origin is used only once per cell cycle.

polymerase-primase; licensing

____ serves as a clamp which is assembled with DNA pol g or epsilon to ensure processivity.

PCNA (proliferating cell nuclear antigen)

RNA primers are synthesized (and extended by pol alpha). The RNA primers are removed by ___ __ and the gaps filled through further DNA synthesis

RNase H

The Okazaki fragments are joined by DNA ___

ligase

The histones and DNA reallocate to form the nucleosome structure. Old histone octamers are not disassembled. Newly synthesized histone octamers associate with only one branch of the replication fork. True or false?

True

An increase in the synthesis of the histones is required for the formation of new nucleosomes during DNA replication. The synthesis of new histones occurs right after DNA replication. True or false?

False; happens simultaneously

New nucleosomes are associated with one of the two daughter strands rather than being distributed between both daughter strands. This has been termed the ___ mechanism for nucleosome synthesis

conservative

How does a cell decide to begin DNA replication (in S-phase)?

proteins called cyclins regulate key steps in cell cycle

Cyclins control ____-____ kinases at various times in cell cycle

cyclin-dependent (CDKs)

Cyclins A and E in particular are synthesized to control the onset of what?

S-phase DNA synthesis by activating kinase CdK2

Cdk2-cyclin E/A complex phosphorylates ___ which causes dissociation this protein which then activates ___

pRb (retinoblastoma protein); E2F

E2F does what?

turns on many genes to activate DNA synthesis such as DNA pol alpha

Inhibitors of DNA replication can block activation of ___

CdKs

Agents which cause DNA damage can be broken down into two main categories: physical agents and chemical agents. Give examples of each

physical agents: high temp, radiation, different wavelengths chemical agents: methylating agents, nitrous acid, nitrosamines, acridine dyes

DNA damaging agents usually act by altering the ___ of DNA and causing disruption of normal ___ bonding of complementary bps

structure; hydrogen

Give the mechanism of a pyrimidine dimer

covalent linkage of two adjacent thymines (could be other pyrimidines) in one of the two polynucleotide chains of DNA

What is a common cause of dimer formation?

UV radiation

What are the consequences of a pyrimidine dimer?

the hydrogen bonding of thymine to their paired adenines is disrupted and results in inhibition of advance of the replication fork

Deamination is a chemically induced or spontaneous loss of an amino group which results in conversion of ___ to ___ or conversion of ____ to _____

cytosine; uracil; adenine; hypoxanthine

Depurination: spontaneous loss of a purine occurs at a rate of about ____ purines per day per cell

10,000 note: this is called an apurinic site in DNA

Single strand breaks are caused by what? Double strand breaks?

single strand: chemical and radiation exposure | double strand: chemical - anticancer drugs

In regards to mechanisms of DNA repair, photo reactivation only operates on what?

pyrimidine dimers

E. Coli enzyme which catalyzes _____ of pyrimidine dimers is called photolyase.

photoreversal

___ is the coenzyme that is involved in photo reversal reaction. Exposure of the cell to visible light is essential for repair to occur.

FADH2

Excision repair is a pathway for removal of ___ ___ modifications of DNA and ____ dimers

bulky chemical; pyrimidine

The first step in excision repair is what?

recognition of damaged dan note: this is followed by "nicking" of dan at the 5' end and 3' end of the damaged region by a repair endonuclease

In regards to excision repair, the damaged region of DNA is removed and replaced with the correct deoxyribonucleoside monophosphates using the undamaged strand as a template. In eukaryotes, DNA polymerase ___ fills in the gap

epsilon

In regards to excision repair, the action action of the DNA ligase is required to form the final ____ bond

phosphodiester

Base excision repair mechanism of the spontaneous deamination of ___ to ___.

cytosine; uracil

Base excision repair does not result in disruption of normal hydrogen bonding and therefore requires the activity of another enzyme system to assists in the repair of this type of modification. What is this enzyme?

uracil dna glycosylase: hydrolyzes the bond between uracil and deoxyribose resulting in removal of uracil from the dna

In regards to base excision repair, when uracil is removed from the dna, this results in an ____ site in the dna and subsequent recognition by a specific ___

apyrimidinic; endonuclease note: after recognition, poly and ligase finish job

In SOS repair, many enzymes are induced in response to high DNA damage levels. DNA synthesis may occur through damaged template region but the result is error prone repair. Opposite a damaged base, repair DNA polymerase inserts an __ hence some of the time a mistake is made

Recombination repair is also involved in the SOS response. The problem is a stalled replication fork due to what?

a thymine dimer

After a pause, DNA poly will skip the block and resume synthesis _____ of the block. The skip is filled in via recombination with the complementary undamaged strand.

downstream note: bacteria use the recA protein to assist in recombination events in this type of repair

Individuals afflicted with this disorder have an increased sensitivity to sunlight, resulting in the development of many skin cancers. This has been shown to be due to defects in the repair of UV-induced damage to dna.

Xeroderma pigmentosum (XP)

XP is inherited in a ___ ____ manner, which is consistent with mutation of a single gene

autosomal recessive

Classical XP defect is thought to be associated with which repair system

excision repair, specifically the endonuclease required to "nick" the damaged DNA strand at the 5' end

How many different genetic loci have been identified of the XP disease

XP variants tend to have mutations in repair DNA poly ___

Ataxia telangiectasia, Faconi's anemia, and Bloom's syndrome are all associated with an increased frequency of cancer. This is due to ____ abnormalities which can be detected in cells from patients who have ___ defects

chromosomal; repair

``` For each of the following diseases, give the sensitivity, type of cancer, and symptoms of each: XP Ataxia telangiectasia Fanconi's anemia Bloom's syndrome Cockayne's syndrome ```

XP: UV radiation; skin carcinomas/melanomas; skin and eye photosensitivity Ataxia telangiectasia: gamma irradiation; lymphomas; ataxia, dilation of blood vessels in skin, chromosome aberrations Fanconi's anemia: cross linking agents; leukemias; hypo plastic pancytopenia, congenital anomalies Bloom's syndrome: UV; leukemias; photosensitivity Cockayne's syndrome: UV; various tumors; neurological defects, dwarfism

patients of this syndrome lack a transcription helicase used in repair during gene transcription

Cockayne's syndrome note: this is known as transcription coupled repair. the mutated CSB protein in CS patients does not allow dan damaged genes to be repaired during transcription. This causes loss of some mRNA production and hence a more severe phenotype of CS compared to XP

It has been shown that loss of DNA repair pathway may underlie tumor formation in HNPCC (hereditary nonpolyposis colorectal cancer). The culprit genes are involved in ____ repair and mutations in these genes would be strong indication that the individual was predisposed to this cancer

mismatch

HNPCC genetic defect is one of the most commonly inherited and accounts for around ___% of CRC cases

BRCA1 and BRCA2 have been linked to hereditary breast and ovarian cancers. These genes code for ___ repair proteins linked to Fanconi's anemia type

recombination