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Advanced Molecular Bio > DNA Replication, Damage, and Repair > Flashcards

DNA Replication, Damage, and Repair Flashcards

1
Q

DNA polymerase

A

Essential enzyme for DNA replication. 2 DNA polymerases work cooperatively in complex. They synthesize DNA in the 5’ to 3’ direction.

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2
Q

Nucleotide triphosphates

A

All nucleotides come into DNA synthesis as triphosphates, but lose 2 phosphates as they are incorporated into the final DNA strand- left with one

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3
Q

Directionality of DNA

A

Synthesized in 5’ to 3’ direction, because new nucleotides are added to the OH at the 3’ end. 5’ and 3’ refer to the carbons on deoxyribonucleoside monophosphate (deoxyribonucleotide) of DNA

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4
Q

RNA primers

A

Synthesized by DNA primase, they are nucleotide (RNA) sequences that are 10-25 nucleotides long. Primers are added to the 5’ end of DNA and provide a 3’ end for DNA polymerase to add nucleotides to. They are necessary for DNA replication and are the first segment added to the new DNA strand. Later, the primers will be removed and replaced with DNA.

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5
Q

Replication fork machinery

A

Has 5’ to 3’ directionality, but the 2 strands are antiparallel to each other (one is 5’ to 3’, the other is 3’ to 5’). Consists of the leading and lagging strands. The replication fork requires 2 DNA polymerase, DNA primase, and DNA ligase. Other proteins are also necessary (DNA helicase, SSB proteins), as well as the sliding clamp and clamp loader

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6
Q

Leading strand

A

Also called the parental strand. It is positioned 3’ to 5’. This allows for smooth DNA synthesis because DNA polymerase can synthesize in the 5’ to 3’ direction

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7
Q

Lagging strand

A

Positioned in the 5’ to 3’ direction, antiparallel to the leading. This means that DNA synthesis by DNA polymerase must be discontinuous. It occurs through a “backstitching” mechanism (DNA polymerase does not move backwards). Achieved by many RNA primers and the synthesis of Okazaki fragments. The fragments are eventually joined by DNA ligase. Each fragment needs a new set of primers

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8
Q

DNA primase

A

Synthesizes complementary short RNA primers (10 nucleotides). New primers are needed for each Okasaki fragment

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9
Q

DNA ligase

A

Seals remaining gap b/w Okazaki fragments & DNA that replaced primer

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10
Q

DNA ligase sealing of Okazaki fragments (5 steps)

A
  1. DNA ligase reacts with ATP. ATP is hydrolyzed to AMP and pyrophosphate
  2. AMP is added to the lysine of DNA ligase, forming a complex
  3. AMP is transferred from the lysine to the 5’ phosphate of nicked DNA
  4. Nucleophilic attack by 3’ OH on activated 5’ pyrophosphate group. This is possible because the second phosphate is a favorable leading group
  5. DNA nick is sealed and AMP is released
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11
Q

DNA helicase

A

An allosteric motor protein that unwinds the DNA double helix. One part of the protein hydrolyzes ATP, resulting in a conformational change in another part of the protein. The conformational changes causes movement of helicase and unwinding of the parental strands

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12
Q

Single strand DNA binding (SSB) proteins

A

They help DNA helicase by stabilizing unwound single stranded DNA, and prevent a strand from base-pairing with itself. Essentially, they act as chaperones for single stranded DNA. SSBs do not cover the bases of nucleotides when they bind to DNA- bases need to remain available for templating

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13
Q

DNA helicase mechanism (5)

A
  1. ATP binding shifts the motor protein, causing a conformational change
  2. The bound ATP is hydrolyzed to ADP and inorganic phosphate (Pi)
  3. ATP hydrolysis changes conformation
  4. Release of ADP and Pi drives the protein back to the original conformation
  5. This process of ATP binding, hydrolysis, and release results in repeated conformational changes and produces a “walking” motion in a cyclical manner
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14
Q

Sliding clamp

A

Keeps DNA Polymerase firmly on DNA as it moves, releases DNA Polymerase once double stranded DNA is encountered

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15
Q

Clamp loader

A

Utilizes ATP hydrolysis to physically load the sliding clamp on to primer-template junction of DNA

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16
Q

Sliding clamp and clamp loader mechanism (6 steps)

A
  1. Energy released from ATP hydrolysis allows the clamp loader to load the sliding clamp around the double helix
  2. Clamp loader releases and the sliding clamp binds to the back of DNA polymerase
  3. Sliding clamp slides as DNA polymerase moves, keeping DNA polymerase bound
  4. On the leading strand, the sliding clamp is tightly bound the entire time
  5. On the lagging strand, the sliding clamp is released once DNA polymerase reaches the preceding Okazaki fragment
  6. DNA polymerase associates with the new sliding clamp on the RNA primer of the next Okazaki fragment
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17
Q

Replication fork proteins

A

Replication fork proteins are a multienzyme replication machine. DNA ligase is not part of this machinery, it works with DNA repair enzymes that operate behind the replication fork

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18
Q

As helicase unwinds DNA, what happens to DNA ahead of the replication fork?

A

The helix ahead of the fork rotates/twists due to pulling

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19
Q

DNA supercoiling

A

DNA is twisted- when DNA is pulled apart, you get supercoils ahead of the replication fork

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20
Q

Positive supercoiling

A

DNA can get twisted so badly that DNA gets “knotted up”. This would cause the stalling of DNA synthesis. However, topoisomerases both prevent and undo positive supercoiling

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21
Q

DNA topoisomerases functions (2)

A
  1. Relieve tension & reduce energy required to unwind replicating chromosome- works to prevent supercoiling
  2. Untangles supercoiled DNA- a problem especially where 2 double helices cross-over (or fold over) 1 another
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22
Q

Topoisomerase 1

A

Actively prevents supercoiling. It creates a transient single-strand nick in the DNA backbone, ahead of the replication fork. The nick allows free rotation by using backbone opposite nick as “swivel”, which prevents supercoiling to some extent. The covalent linkage that joins topoisomerase to DNA phosphate retains energy of cleaved phosphodiester bond, allowing rapid resealing w/ no additional energy cost

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23
Q

Topoisomerase 2 (DNA gyrase)

A

Undoes supercoiling that has already occurred. Covalently links to both DNA strands, creating double strand break and undoing the knot. Operates when 2 double helices cross one another

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24
Q

DNA topoisomerases

A

Can be thought of as reversible nucleases

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25
Q

How does DNA replication begin?

A

Begins at the replication origin (OriC in prokaryotes)- sequences in the template DNA that attract initiator proteins. This sequence is A-T rich, which is especially easy to open. This is because there are 2 hydrogen bonds between AT and 3 between GC.

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26
Q

Initiator proteins

A

Recognize the replication origins: DNA designation in prokaryotes and origin recognition complex (ORC) in eukaryotes. Pry apart H-bonds between bases- this allows helicase to hop onto ssDNA

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27
Q

Initiation of replication in prokaryotes and eukaryotes

A

In prokaryotes, there is one origin of replication per chromosome. However, in eukaryotes, there are many origins of replication, called replication units (clusters). They can have 20-80 origins spaced between 30,000-250,000 nucleotides from each other

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28
Q

Initiation of DNA replication in prokaryotes (4)

A
  1. Oligomer of ~20 initiator DnaA proteins binds oriC
  2. HU protein aids in the separation of the A-T rich region in an ATP-dependent process (45 bp open complex)
  3. DnaB (another name for helicase in bacteria- also an initiator protein) binds A-T rich region with help of DnaC (prepriming complex)
  4. Makes primers, DNA polymerase can begin making DNA
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29
Q

Initiation of DNA replication in eukaryotes

A

In contrast to bacteria, which replicates DNA continually, DNA replication only occurs during DNA synthesis phase (S phase) of cell cycle. Lasts around 8 hours for mammals. By the end of the S phase, each chromosome replicates to produce 2 copies. Copies remain joined at centromeres until the mitosis (M) phase

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30
Q

Eukaryotic sections of the origins of replication (3)

A
  1. ORC binding site
  2. Unwinding site
  3. Abf1-binding site
31
Q

ORC binding site

A

Binding site for the origin recognition complex, which are the initiator proteins in eukaryotes

32
Q

Unwinding site

A

A-T rich region

33
Q

Abf1 binding site

A

Binding site for auxiliary proteins that recruit ORC

34
Q

Pre-replication complex (Pre-RC)

A

Consists of several ORCs bound to the eukaryotic origin of replication, and other proteins. Forms first before recruiting DNA polymerase, helicase, etc

35
Q

Initiation of DNA replication in eukaryotes (6 steps)

A
  1. ORC-origin interaction persists through the entire cell cycle. The interaction begins in G1
  2. Helicase-loading proteins (CDC 6, CDT 1) associate with ORC in G1
  3. Cdc6 & Cdt1 recruit helicase to ORC in G1
  4. Cyclin-dependent kinases (Cdks) phosphorylate Cdc6 and ORC between G1 & S phases
  5. Helicase is now free to act, replication occurs in S phase
  6. Following G2 and M phases, ORC dissociates allowing assembly of new pre-RC
36
Q

Cell cycle phases

A
  1. Interphase- G1, S, G2
  2. Mitotic (M) phase
37
Q

Is euchromatin or heterochromatin replicated first?

A

Euchromatin. Heterochromatin replication is initiated toward the end of the S phase since it takes longer to be unwound. This acts as an extra measure of regulation

38
Q

Nucleosomes during replication

A

There are many redundant genes for histones- histones are produced at a mass that equals new DNA synthesis. This is because we must have enough histones to form all of the nucleosomes, as DNA is essentially doubled in the cell during S phase. Histones are mainly synthesized in the S phase, giving them a boost in quantity. They are not produced during interphase like most proteins. The rate of histone mRNA increases 50 fold due to increased transcription

39
Q

As the replication fork proceeds, what happens to the histones of the nucleosomes? (4 steps)

A
  1. H3-H4 tetramer stays bound transiently, distributed randomly to daughter strands
  2. H2A-H2B dimers released
  3. New H3-H4 fills in empty spaces
  4. H2A-H2B (old & new) completes assembly of the nucleosome. A lot of nucleosomes are a mixture of old and new histones
40
Q

Nucleosome assembly during replication

A

Some histones are completely new, some are completely old (left over from the parental strand), most are a combination

41
Q

Regulation of initiation of replication

A

Replication is highly regulated. The only point at which bacteria can control DNA synthesis is at initiation. Once replication begins, we’re committed to it. Lack of methylation of A acts as a signal that DNA has just been replicated and new DNA should not be made. This is called the refractory period.

42
Q

How does DNA replication end in prokaryotes?

A

Prokaryotes have termination regions called Ter in their circular chromosomes. They prevent DNA replication in a particular direction (can be clockwise or counterclockwise). Terminator utilization substance (Tus) proteins binds Ter and acts like a wall. Once replication machinery hits this “wall”, it falls apart, and replication terminates

43
Q

Ter sequences in E. coli (6)

A

TerA, TerD, & TerE prevent counterclockwise replication; TerC, TerB, & TerF prevent clockwise

44
Q

How does DNA replication end in eukaryotes?

A

There are telomeres at the ends of linear DNA, closing the ends of the chromosomes. Telomeres are created by telomerase

45
Q

Telomeres

A

The protruding 3’ and single stranded end of the chromosome tucks into the double stranded DNA of telomeric repeats. It forms a t-loop. When DNA from more than one source comes together, it is called a heteroduplex

46
Q

Holliday junction

A

A critical structural during DNA recombination, a similar structure is found in telomeres

47
Q

End replication problem

A

Due to the nature of lagging strand synthesis where telomeres aren’t replicated, the chromosome ends (telomeres) get shorter and shorter w/ each replication. Eventually, the telomeres would be so short that the cells wouldn’t be able to replicate. Solved by telomerase

48
Q

Telomerase

A

Has RNA template & reverse transcriptase activity to extend the 3’ end of the parental DNA strand. Uses the RNA template to synthesize DNA. It is responsible for creating a full telomere and solving the end replication problem.

49
Q

Telomerase mechanism (4)

A
  1. Synthesis of telomeres results in protruding 3’ end (elongated). Telomerase recognizes telomeric repeats here
  2. Uses reverse transcription to replenish the telomere on the parental strand
  3. DNA Pol takes over and synthesizes the telomere on the lagging strand
  4. Protruding 3’ end tucks into dsDNA of telomeric repeats: t-loop
50
Q

Which sequence does telomerase recognize?

A

Recognizes telomere repeats (GGGTTA) at tip of chromosomes on the parental DNA strand

51
Q

Which cells have low telomerase activity?

A

Most somatic cells- this gives them a finite number of replications

52
Q

Which cells have high telomerase activity?

A

Cells that need constant renewal – those of bone marrow or skin, activated lymphocytes, and stem cells. These cells retain their high telomerase activity, which allows them to have a large number of replications. Cancer cells also have high telomerase activity- they will never have a problem with shortening telomeres

53
Q

Spontaneous DNA damage

A

Most DNA damage is spontaneous. Types of spontaneous damage includes oxidative damage (ROS), hydrolysis, and methylation (adding of a methyl group)

54
Q

Most frequent DNA damage (2 types)

A
  1. Depurination- removing a purine base from a nucleotide
  2. Deamination- removing an amine (NH3) group from a nucleotide.
    Both forms are hydrolytic damage- water drives the reactions
55
Q

Pyrimidine (thymine) dimers

A

Frequently occurs among neighboring thymines due to photochemical damage from UV radiation. Two pyrimidines (usually thymines, but can be cytosine) are cross-linked. Forms a large, bulky lesion in the DNA, which halts replication and transcription

56
Q

Base excision repair

A

Used when a defective base is present. DNA glycosylases recognize the altered bases and remove them by hydrolysis. AP endonucleases are also involved

57
Q

DNA glycosylases

A

An enzyme that recognizes altered bases and repairs DNA. The enzyme recognizes the damaged bases because they are “flipped out” from the double helix.

58
Q

AP endonuclease

A

Removes the phosphodiester backbone

59
Q

Base-excision repair mechanism (5)

A
  1. The base is “flipped” out from the double helix and is recognized by DNA glycosylases
  2. DNA glycosylase removes the damaged base
  3. AP endonuclease 1 removes the piece of the phosphodiester backbone
  4. Protein XRCC1 serves as a scaffold. DNA polymerase comes in and replaces the removed nucleotide
  5. DNA ligase seals the remaining gap, fully repairing the DNA
60
Q

Long-patch repair

A

Acts as a backup to base excision repair. Used in situations where the phosphodiester backbone is resistant to AP endonuclease, as with X-ray damage

61
Q

Long-patch repair mechanism (5)

A
  1. ssDNA break due to X-ray
  2. XRCC1 scaffold protein binds. It recruits the DNA repair enzyme PARP. Both proteins protect DNA ends
  3. The PCNA scaffold protein binds and “lifts” the strand so DNA Polymerase can bind. It uses the bottom strand as a template and extends several nucleotides- doing more than just replacing the one base
  4. Endonuclease removes the extra “patch” of nucleotides
  5. Ligase seals, several bases are replaced
62
Q

Types of DNA repair (4)

A
  1. Base excision repair
  2. Long-patch repair
  3. Nucleotide excision repair
  4. Double strand break repair
63
Q

Nucleotide excision repair

A

Used to repair bulky lesions (pyrimidine dimers, covalent binding of bases with hydrocarbons). In fact, this is the primary means by which pyrimidine dimers are repaired. Serious diseases can result from a defect in this mechanism. Large, multienzyme complex recognizes distortion in double helix and cleaves

64
Q

Mechanism of nucleotide excision repair (4)

A
  1. Excision nuclease recognizes the DNA lesion
  2. Makes two cuts, one several bases upstream from the lesion and one several bases downstream
  3. DNA helicase unzips and removes the cut segment of the genome
  4. DNA polymerase repairs the stretch of DNA, and DNA ligase seals the gaps
65
Q

Xeroderma pigmentosum

A

A genetic disease where patients lack nucleotide excision repair. As pyrimidine dimers can occur quite often, it is problematic if they can’t be adequately repaired. These individuals are very sensitive to UV light

66
Q

Melanoma

A

The more pyrimidine dimers you get, the more you are predisposed to melanoma. Persistent UV exposure can lead to a defect in nucleotide excision repair

67
Q

Double strand break repair

A

Double strand breaks in DNA can be caused by ionizing radiation, replication errors, and oxidizing agents. Can be repaired by nonhomologous end joining or homologous recombination

68
Q

Nonhomologous end joining

A

The broken ends of the DNA are brought together by DNA ligation. There may be a loss of nucleotides due to degradation from the DNA ends. Therefore, there can be DNA overhangs (blunt ends). DNA ligase comes in and glues the pieces together. This process isn’t very efficient process for genes due to nucleotide loss. However, it can be acceptable as only a small fraction of the mammalian genome codes for proteins

69
Q

Lagging strand synthesis

A

Requires DNA primase to make a new primer for each Okazaki fragment. However, the primers do not remain, the DNA repair system removes the primers and fills in DNA. DNA ligase will then seal the remaining gaps between Okazaki fragments

70
Q

DNA helicase structure

A

It is a helical structure with a central pore. The central pore is big enough for a single strand of DNA, which is how it works to separate the 2 strands

71
Q

Protection of telomeres 1 protein (POT1)

A

Binds to the G-rich, single stranded 3’ overhang. It stabilizes the T loop. POT1 also suppresses DNA repair enzymes that might otherwise recognize the structure as DNA damage

72
Q

Telomerase structure

A

Telomerase contains an enzymatic portion that carries out reverse transcription. It also has its own mRNA template that it reverse transcribes to make telomeres out of DNA

73
Q

Deamination of cytosine

A

Creates uracil- hydrolytic reaction where ammonia is released and O is added

Advanced Molecular Bio (13 decks)

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