UF BCH4024 Exam 4: DNA Replication

in which direction does polynucleotide synthesis occur & what molecule serves as the substrate?

5'→3' & nucleotide triphosphates act as substrates

what is the role of DNA polymerase in polynucleotide synthesis & what does it require to accomplish this role?

synthesizes new DNA strands by adding one dNMP (deoxyribonucleoside monophosphate) to the 3' end of a DNA chain

requires DNA template chain & a primer

what is the role of RNA polymerase in polynucleotide synthesis & what does it require to accomplish this role?

synthesizes new RNA strands by adding one NMP (ribonucleoside monophosphate) to the 3' end of an RNA chain

requires DNA template chain

what is the role of reverse transcriptase in polynucleotide synthesis & what does it require to accomplish this role?

synthesizes new DNA strands from RNA by adding one dNMP to the 3' and of a DNA chain

requires RNA template & a primer

what does exonuclease do?

breaks a phosphodiester bond at one terminal of a polynucleotide chain (can go 5'→3' OR 3'→5')

what does endonuclease do? what are two important characteristics of this enzyme?

breaks a phosphodiester bond within a polynucleotide chain

1. can be sequence-independent or sequence-dependent

2. can break a single-strand (nick) or a double-strand

what does excinuclease do?

breaks two phosphodiester bonds within a single polynucleotide chain

define palindromic sequences:

both strands of DNA have the same sequence when read 5'→3'

what are restriction endonucleases/enzymes?

sequence-specific endonucleases that only break phosphodiester bonds at restriction sites

what are restriction sites?

short palindromic sequences that can be broken by restriction endonucleases/enzymes

what are sticky ends, how are they formed, & why are they useful?

unpaired bases formed by restriction endonuclease cleavage of a palindromic sequence that can then base pair by forming hydrogen bonds to form new DNA sequences that might otherwise not exist in nature

what does DNA ligase do? what energy source does it require?

links 2 existing DNA chains by forming a phosphodiester bond resulting in a continuous 5'→3' DNA strand

requires AMP (from ATP or NAD+ in humans)

what is the primary role of DNA ligase in vivo?

seals single-strand breaks (AKA nicks)

what is recombinant DNA & how is it formed?

new combinations of sequences formed by DNA ligase joining blunt-ends or sticky ends created by endonucleases (AKA restriction enzymes)

what does semi-conservative replication mean?

DNA replication will yield 2 DNA molecules, each with one parental & one synthesized strand

in what experiment was the semi-conservative theory of DNA replication proved?

Meselson & Stahl experiment

in the Meselson & Stahl experiment what was used as the:

1. 2 types of mediums

2. chemical gradient

3. cell genome

1. normal (14N) & heavy (15N)

2. CsCl

3. e. coli

what is the role of DNA polymerase & what does it require?

catalyzes DNA synthesis one nucleoside monophosphate at a time in the 5'→3' direction.

requires:

1. DNA template strand

2. DNA or RNA primer annealed to template strand

3. 3'-OH on the primer

4. dNTP substrates

what regulatory process do all DNA polymerases possess to prevent replication errors & what would happen if there was an error in replication?

the active site of DNA polymerase restricts base pairing to Watson-Crick-Franklin base pairs in a regulatory process known as pre-synthetic error control. errors in replication would introduce a permanent mutation into the genome that would be inherited by daughter cells

what mechanism, besides pre-synthetic error control, do high-fidelity DNA polymerases utilize to further prevent permanent mutations within the genome?

proofreading: there are 2 active sites within high-fidelity DNA polymerases which are the catalytic site for DNA synthesis & a 3'→5' exonuclease site for removing mis-incorporated nucleotides

is DNA polymerase I or DNA polymerase III a better regulator for our genome & why?

DNA polymerase III

faster polymerization rate & faster processivity rate

what is processivity as it relates to DNA polymerase?

the number of nucleotides that can be added to the polymerase before dissociation

which two DNA polymerases are involved in regulating our genomes?

DNA polymerase I & DNA polymerase III

what regulatory feature does DNA polymerase I possess that DNA polymerase III lacks?

5'→3' exonuclease activity (DNA polymerase III only exhibits 3'→5' exonuclease proofreading)

what is the replication fork?

where the parent DNA is being used as a template for replication by DNA polymerase

what does helicase do?

unwinds DNA strands by breaking H-bonds just ahead of the DNA polymerase

leading strand vs. lagging strand

leading: DNA synthesis occurs continuously from 5'→3'

lagging: DNA synthesis occurs discontinuously in Okazaki fragments as a series of 5'→3' reactions

what is the regulated step that controls DNA synthesis?

initiation

what are the 2 primary reasons why DNA synthesis is highly regulated? (other than mutations)

1. too much or too little DNA is bad for the cell

2. DNA replication is energy (ATP) expensive

what is the origin of replication in bacterial DNA synthesis?

oriC

what is the first protein involved in bacterial DNA replication?

DnaA

what is the DNA unwinding element (DUE)?

an AT-rich segment where strand separation occurs

what is the mechanism of DNA methylation in regulating the initiation of e. coli DNA replication?

1. the N⁶ position of adenine within the 5' position of oriC is methylated by Dam methylase (DNA adenine methylation)

2. once replication is complete, DNA is hemimethylated, meaning the parent strand is methylated while the synthesized strand is not

3. the hemimethylated oriC becomes a binding site for certain proteins within the plasma membrane. once the proteins are bound to the oriC initiation site, it is sequestered to the membrane, preventing anything else from binding to it to prevent the initiation of further replication

4. after a time, the DNA is released from the plasma membrane & fully methylated so replication can initiate again

what is the core enzyme & accessory subunits of DNA III Holoenzyme?

core = DNA polymerase III

accessory subunits = clamp loader & β clamp

why is an RNA polymerase (primase) needed to prime the DNA template before DNA polymerase III can bind to it in e. coli DNA replication?

unlike DNA polymerase, RNA polymerase can synthesize RNA de novo (meaning without a primer), so it can take the first 2 nucleotides into its catalytic site to form a phosphodiester bond between them and then start adding nucleotides to the 3' end. DNA polymerase can't be used for priming the template because it can only add nucleotides to an existing 3' end

what are the functions of the clamp loader subunit of the DNA polymerase III holoenzyme?

1. serves as a scaffold for DNA polymerase III complex

2. assembles the β clamp onto DNA using ATP

3. coordinates the replication fork by facilitating protein-protein interactions with DnaB helicase through Τ subunits

what is the purpose of the β clamp (sliding clamp) subunit of DNA polymerase III holoenzyme?

it tethers the core & encircles the DNA, sliding back and forth freely to decrease the polymerase dissociation from DNA, ultimately increasing processivity

what is the function of topoisomerase?

it adds/removes supercoils in DNA

what does SSB (single strand binding protein) protect single-stranded DNA from after helicase separates the strands?

nucleases, reannealing, & secondary structure formation

what are the 4 stages of eukaryotic cell division?

1. G1

2. S

3. G2

4. mitosis

eukaryotic cell division is primarily regulated by which two molecules?

cyclins & CDKs

in adults, eukaryotic tissues stop dividing and enter which phase?

G0

what type of cells continue dividing in adulthood?

stem cells

the actual act of eukaryotic cell division occurs in which phase?

S

in eukaryotic cells, an origin of replication will usually be where? and are usually associated with what?

AT-rich element & genes

what does licensing refer to in mammalian cells?

in which phase of the cell cycle does licensing begin? what 3 preparatory processes occur?

G1

1. ORCs (origin of replication complexes) bind tightly to origin of replication sequences in DNA

2. CDC6 (cell division cycle 6) & CDT1 join & load 2 helicase MCM (Mini Chromosome Maintenance) molecules

3. MCM translocates 3'→5' along the leading strand template using ATP

in which phase of the cell cycle does DNA synthesis begin? what processes occur in this phase to initiate synthesis?

S

replication is initiated by phosphorylation of cyclin-CDK,

the replisome is assembled & bidirectional synthesis is initiated in all origins of replication

once cyclin-CDK is activated by phosphorylation in the S phase & replication begins, what phase is inhibited to ensure that origins of replication are only used once per round of replication?

G1

what enzyme is the helicase in the eukaryotic replisome?

MCM

what eukaryotic enzyme synthesizes the leading strand? what are its two associated characteristics?

DNA polymerase ε

high processivity & proofreading

what eukaryotic enzyme synthesizes the lagging strand? what regulatory process is it involved in?

DNA polymerase δ

proofreading

what is DNA polymerase α-primase?

complex containing primase for RNA primer synthesis & a separate DNA synthesis activity in eukaryotic DNA replication

what is the clamp loader called in eukaryotes?

RFC (replication factor C)

what is the sliding clamp called in eukaryotes?

PCNA

what is RPA (replication protein A)?

single-strand DNA binding protein (SSB) in eukaryotes

what is the big difference between replication elongation in prokaryotes & eukaryotes?

prokaryotes have one enzyme (DNA polymerase III) for both the leading & lagging strands, while eukaryotes have one enzyme for each strand; DNA polymerase ε for the leading strand & DNA polymerase δ for the lagging strand

which eukaryotic enzyme is also referred to as the "strand-displacing" DNA polymerase?

DNA polymerase δ

what does FEN1 do?

flap endonuclease 1 breaks the phosphodiester bond between the RNA primer "flap" structure on the lagging strand, that was displaced by DNA polymerase δ, & and the DNA chain

in both prokaryotes & eukaryotes what type of bond must be formed between Okazaki fragments to form a complete strand? what enzyme does this?

phosphodiester bond & DNA ligase

which strand in a telomere is shorter?

the complimentary (CA) strand

what is the T loop in telomeres & what is its function?

a specialized structure that sequesters the single-stranded end of the telomere by base pairing; it protects the 3' ends of the chromosome from nucleases & other enzymes that repair double-strand breaks

which protein in the T loop protect the ends of the single-stranded 3' end within a DNA duplex?

shelterin (TRF1 & TRF2)

what structures within eukaryotic somatic cells keep track of the cells "age" by shortening and prevent the loss of important DNA & development of cancer?

telemores

what is the senescence state & when does it occur?

permanent G0, occurs when telomeres get too short to safely divide

which two types of eukaryotic cells do not undergo telomere shortening & why?

germ & stem cells because they're young & undifferentiated

what enzyme replicates telomeres and how does it do so?

telomerase, a specialized reverse transcriptase that replicates the ends of chromosomes to lengthen telomeres

in which cells is telomerase expressed in & how does it work?

germ (sperm, eggs, & embryonic cells) & stem cells; telomerase binds to the 3' overhang where it base pairs its RNA template to the 3' end adds nucleotide base pairs, synthesizing a TG-rich strand

which strand is lengthened by telomerase & what enzyme "fills in" the complimentary strand?

the TG strand is lengthened & the CA strand is filled in by DNA polymerase α-primase

why don't mature eukaryotic cells express telomerase?

to keep track of the cell's age & prevent cancer

how can cancer cells express telomerase?

they can reactivate their telomerase gene that is supposed to be inhibited, resulting in proliferation by evading senescence & apoptosis pathways