Chapter 12: DNA Replication

Briefly describe the characteristics and functions of the different DNA polymerases in E. coli. Include functions that all of them have in common

(DP = DNA polymerase) - there are 5 total Has 5'→3' polymerase activity: - DP I - DP III - DP IV - DP V Has 3'→5' polymerase activity: - NONE Has 5'→3' exonuclease activity: - DP I only Has 3'→5' exonuclease activity: - DP I - DP II - DP III FUNCTIONS OF EACH - DP I: removes and replaces primers - DP II: DNA repair; restarts replication after damaged DNA halts synthesis - DP III: elongates DNA - DP IV: DNA repair - DP V: DNA repair; translesion DNA synthesis All 5 DNA polymerases.... - synthesize any sequence specified by template - 5'→3' polymerization - use dNTPs to synthesize new DNA - require a 3' OH group to initiate synthesis - catalyze the formation of phoshodiester bonds - as associated with a number of proteins

What are the different types of topoisomerases?

(recall that topoisomerases control supercoiling by breaking and reforming bonds in the strands) Type I topoisomerases create single-strand breaks, and type II create double-strand breaks.

describe initiation stage and the unwinding stage of bacterial DNA replication

*Initiator proteins* bind to the DNA molecule's origin of replication (it only has one) and causes a short section of the DNA to unwind (this marks the end of initiation). ...This allows *DNA helicase* (which can only bind to single-stranded DNA) to bind to the lagging strand TEMPLATE and it begins unwinding, moving the replication fork in the 5'→ 3' direction by breaking the hydrogen bonds between bases. As DNA unwinds via DNA helicase, a tetramer of *single-stranded-binding proteins* (SSBs) attach to the exposed single-stranded template strands (SSBs can bind to any single-stranded DNA regardless of its base sequence). Each tetramer covers 35-65 nucleotides to protect the single-stranded chains against the formation of secondary structures (eg hairpins) that would interfere with replication. *DNA gyrase* is a type II topoisomerase that reduces the torque that builds up ahead of the replication fork by breaking and reforming segments of double-stranded DNA. This action requires ATP.

Explain the processes that cause bacterial DNA replication to have such a low rate of error

*nucleotide selection* by DNA polymerases is vary particular, and errors in this process arise only about once per 100,000 nucleotides. Most of the errors that do arise in nucleotide selection are corrected in a second process called *proofreading.* When a DNA polymerase inserts an incorrect nucleotide into the growing strand, the 3' OH group of the mispaired nucleotide is not correctly positioned in the active site of the DNA polymerase for accepting the next nucleotide. The incorrect positioning stalls the polymerization, and the 3'→5' exonuclease activity of the DNA polymerase removes it, then inserts the correct nucleotide. A 3rd process, called *mismatch repair* corrects errors after replication is complete. a mismatched nucleotide will cause a deformity in the secondary structure of the DNA, which will be recognized by enzymes. The enzymes use the original nucleotide strand to fix this, so the enzymes must be able to distinguish the old DNA from the new. Methylation of the DNA strands AFTER replication makes this possible. Immediately after synthesis, the new strand is not yet methylated, but the old strand still remains methylated from before.

In comparison with prokaryotes, what are some differences in the genome structure of eukaryotic cells that affect how replication takes place?

- the larger size of the eukaryotic genome - the linear structure of eukaryotic chromosomes - the association of eukaryotic DNA with histone proteins

what are some diseases associated with abnormalities of telomere replication?

- werner syndrome - deficiency of en enzyme necessary for the replication of telomeres. Signs of premature aging begins in adolescence - Dyskeratosis congenita (DKC) - bone marrow cant produce enough new cells - cancer - telomerase is expressed in 90% of all cancers. Cause is unclear, but you do not want telomerase present in somatic cells because it promotes cancer by causing indefinite cell proliferation.

The components of replication can be combined into what 3 major groups?

1. a template consisting of single-stranded DNA 2. raw materials (substrates) to be assembles into a new nucleotide strand 3. enzymes and other proteins that "read" the template and assemble the substrates into a DNA molecule

nucleotides are added to the ____ end of a growing chain

3'

replication takes place in ___ stages:

4 stages: initiation, unwinding, elongation, termination

you should always read a sequence in the ____ direction

5'→3'

describe the other 2 models that were initially proposed for DNA replication before semiconservative replication was accepted as the true model

Conservative replication- the entire double stranded DNA molecule serves as a template for a whole new molecule, and the original DNA molecule is conserved during replication dispersive replication- both original nucleotide strands break down into fragments that serve as templates for the synthesis of new DNA fragments which reassemble into 2 new DNA molecules made of fragments of old and new DNA - none of the original molecules is conserved.

describe elongation in eukaryotic nuclear DNA replication

DNA polymerase α has primase activity which initiates DNA synthesis by synthesizing an RNA primer, followed by a short string of DNA nucleotides. Then, DP δ completes replication on the lagging strand. DP ε replicates the leading strand.

discuss the occurrence of errors in eukaryotic DNA synthesis

DNA polymerases α, δ, and ε are capable or replicating fast and with high fidelity because they have active site that snugly and exclusively accomodate all 4 normal DNA nucleotides. As a result, distorted DNA templates and abnormal bases are not often accomodated within the active sites. when these errors occur, these high-fidelity DNA polymerases stall and are unable to bypass the lesion. Other DNA polymerases have lower fidelity, but are able to bypass distortions in the DNA template. These specialized *translesion DNA polymerases* generally have a more open active site and can accomodate and copy templates with abnormal bases, distorted structures, and bulky lesions. Thus, they can bypass these errors. In the case of a stalled high-fidelity DNA polymerase, these lower fidelity DP's will take over, bypass the lesion, and continue replicating a short section of DNA. Then they detach from this strand and the high-fidelity DP's resume replication. Errors produced by the translesion DNA polymerases are often repaired by enzymes.

What three DNA polymerases carry out most of the nuclear DNA synthesis during replication in eukaryotes?

DP α, DP δ, DP ε

Describe the unwinding stage of DNA replication in eukaryotes compared to bacteria

Helicases, single-stranded-binding proteins, and topoisomerases (which have a function equivalent to the DNA gyrase in bacterial cells) have been isolated from eukaryotic cells and are assumed to function in unwinding eukaryotic DNA in much of the same way as their bacterial counterparts do

Prokaryotes have one origin of replication per chromosome whereas eukaryotes have many. What differences in DNA replication arise as a result of this difference?

In Eukaryones, a cell must ensure that replication is initiated at thousands of replication origins only once per cell cycle. This is accomplished by the separation of the initiation of replication into two steps: In the first step, the origins are *licenced* - approved for replication. This step takes in G1 of interphase of the cell cycle when *replication licensing factors* (including the origin recognition complex) attach to each origin, allowing a complex called MCM2-7 to bind to an origin. (MCM stands for minichromosome maintenance) The second step occurs in the S phase. The MCM2-7 associates with several cofactors and forms an active helicase which unwinds double stranded DNA for replication.

Explain the end replication problem?

In circular chromosomal DNA replication, elongation around the circle eventually provides a 3' OH immediately in front of the primer. after the primer has been removed, the replacement DNA nucleotides can be added to this OH group. In linear chromosomes with multiple origins, adjacent replicons elongate until they run in the primer from the next origin of replication, and once the RNA nucleotides in the primer are all replaced, it provides the 3' OH necessary to make the two adjacent replicons continuous. But at the very end of a linear chromosome, there is no adjacent stretch to provide this crucial 3' OH. When the terminal (5') primer at the end of a chromosome has been removed, it cannot be replaced with nucleotides, so its removal creates a gap at the end of the chromosome, suggesting that the chromosome should become progressively shorter with each round of replication. This is termed the *end replication problem*. Eukaryotic chromosomes actually shorten much faster than would be expected if all it was shortened by was the 10 or so nucleotides of the primer. But now research demonstrages that the terminal primer is positioned not at the end of a chromosome, but rather 70-100 nucleotides from the complementary 3' end. This means that 70-100 chromosomes are are not replicated during division of somatic cells, and the chromosome shortens by this amount each time. So this explains the end-replication problem. Its important to note that this shorening does not occur in single-celled eukaryotes, germ cells, or early embryonic cells.

Describe termination in bacterial DNA replication

In some DNA molecules, termination occurs when two forks meet. In others, there are specific termination sequences that a termination protein, called Tus in E. coli, binds to to block the movement of helicase, thus stopping the unwinding

Describe the processivity of DNA polymerase III in bacteria

It has high processivity, meaning it is capable of adding many nucleotides to the chain without releasing the template - it usually holds on until replication of the template is complete. This high processivity is ensured by one of the polypeptides that constitutes the enzyme. This ring-shaped polypeptide called the *β sliding clamp* which helps the DNA polymerase slide easily along the template strand during replication.

In bacterial DNA replication, two units of DNA polymerase III are connected - one polymerizing the leading strand and the other polymerizing the lagging strand. How does this happen if nucleotides can only be added in the 5'→3' direction?

Lagging strand loops around

In rapidly dividing bacteria, DNA replication is continuous. How is DNA replication in eukaryotic cells different?

Our DNA replication is coordinated with the cell cycle which is controlled by checkpoints. the G1/S checkpoint holds the cell in G1 until its DNA is ready to be replicated. This system ensures that the DNA is not replicated again right after mitosis

What is rolling circle replication?

Rolling circle replication takes place in some viruses and in the F factor ecoli circular DNA, and is initiated by a break in one of the strands - new nucleotides are then added to the 3' end of the broken strand, using the inner (unbroken) strand as a template.

Describe the elongation stage in bacterial DNA replication

Synthesis of primer: All DNA polymerases requires more than just a bare template strand to begin DNA synthesis - it needs a nucleotide with a free 3' OH group to which the first new nucleotide can be added. RNA polymerases, however, do not require a preexisting 3' OH group to start synthesizing a new strand. *Primase* is an RNA polymerase that synthesizes a short strand (10-12 nucleotides long) of RNA nucleotides called a *primer* which provides the 3' OH group for DNA polymerase to add the first DNA nucleotide to. (RNA primers are later replaced with DNA nucleotides). Since the leading strand replicates continuously, only one initial primer is needed to begin its replication. However a primer is required at the beginning of every Okazaki fragment of the lagging strand, so primase forms a complex with helicase at the replication fork and moves along the template of the lagging strand, ready to lay down new primers. Synthesis of DNA: E. coli have at least 5 DNA polymerases that have been well studied. *DNA polymerase III* acts as the main workhorse of replication - it synthesizes DNA by adding nucleotides to the 3' end of a growing chain. It has two enzymatic activities. Its 5'→3' polymerase activity allows it to add new nucleotides in the 5'→3' direction. Its 3'→5' exonuclease activity allows it to remove nucleotides in the 3'→5' (reverse) direction, enabling it to correct errors. Its basically like a keyboard that can backspace any typos, and then resume typing. like DNA polymerase III, *DNA polymerase I* also has the 5'→3' polymerase activity and 3'→5' exonuclease activity, but in addition, it has 5'→3' exonuclease activity which serves to remove the primers laid down by primase and then replace them with DNA nucleotides in the 5'→3' direction. After DNA polymerase III initiates DNA synthesis and moves downstream, DNA polymerase I replaces the primer with DNA nucleotides. Once DNA polymerase I replaces the last remaining RNA nucleotide in a primer, there still remains a break between the two okazaki fragments. This break, or "nick" is sealed by the enzyme *DNA ligase* which catalyzes the formation of a phosphodiester bond.

Why dont chromosomes in germ cells or early embryonic cells shorten with every replication like they do in somatic cells?

The end of a double-stranded DNA molecule contains a 3' end and a 5' end. The 5' end is the end that shortens by 70-100 nucleotides during replication. The end of a telomere possesses the presence of many copies of a short repeated sequence, which in humans, is 5'-TTAGG-3', followed by a G rich repeat that makes the G-rich 3' overhang. This overhang can be extended by *telomerase.* Telomerase has an RNA component 15-22 nucleotides that are complementary to the G-rich 3' overhang. This RNA sequence pairs with the the overhang, providing a template that then serves to elongate the overhang. The removal of this RNA polymerase allows the 5' end to elongate a little further. Telomerase is only present in single-celled euaryotes, germ cells, embryonic cells, and certain proliferative somatic cells such as bone marrow, all of which must undergo continuous cell division.

Explain the basic mechanism of how new nucleotides are added to a growing nucleotide strand in DNA synthesis

The raw materials used to synthesize a DNA molecule are dNTPs (deoxyribonucleoside triphosphate) which is a nucleoside attached to THREE phosphate groups. In DNA synthesis, the 3' OH group of the last nucleotide on the strand attacks the 5' phosphate group of the incoming dNTP, kicking off the other two phosphate groups on the dNTP, and a phosphodiester bond is created between the two nucleotides. This process is done via a group of enzymes called DNA polymerases. DNA polymerases can only add nucleotides to the 3' end of the growing strand, so new DNA strands always elongate in the same 5' → 3' direction. As the DNA unwinds, the template strand that is exposed in the 3'→5' direction allows the new strand to undergo continuous replication in the 5'→3' direction (elongation proceeds in the same direction the replication fork is moving in). This is the leading strand. The other template strand is exposed in the 5'→3' direction, so after a short length of DNA has been unwound, synthesis must proceed 5'→3'; that is, in the direction opposite that of the replication fork. This is the lagging strand, and the short lengths of DNA produced by this discontinuous replication of the lagging strand are called Okazaki fragments. In bacteria, Okazaki fragments are about 1000-2000 nucleotides long, but in humans they are about 100-200 nucleotides long.

What differences between bacterial and eukaryotic DNA replication arise as a result of the location where it takes place in the cell?

There is evidence that replication in eukaryotes takes place at fixed sites in the nucleus, called replication factories. This suggests that DNA polymerase is fixed and template DNA is threaded through it.

Explain nucleosome assembly

This occurs in eukaryotic DNA but not bacterial DNA. Chromatin structure is disrupted by the replication fork, but nucleosomes are quickly reassembled on the two new double-stranded DNA molecules. Assemmbly requires 3 steps: 1. disruption of the original nucleosomes on the parental DNA molecule ahead of the replication fork 2. the redistribution of preexisting histones on the new DNA molecules 3. the addition of newly synthesized histones to complete the formation of new nucleosomes. Newly assembled octamers contain a mixture of old and new histones. (this was determined in isotope-labeling experiments) Reassembly of nucleosomes during replication is facilitated by proteins called histone chaperones, which are associated with helicase. The histone chaperones accept old histones from the original DNA and new histones and deposit them on to the new DNA molecules. The original histone octamer is broken down into two H2A-H2B dimers and a single H3-H4 tetramer, and the old H3-H4 tetramer is then transferred randomly to one of the new DNA molecules, and serves as a foundation into which the either new or old H2A-H2B dimers are added. Some Octamers consists all new histones, the assembly of which is facilitated by the protein called chromoatin-assemble factor

discontinuous replication is a result of which property of DNA?

antiparallel nucleotide strands

What is meant by semiconservitave replication?

each of the original nucleotide strands from the original double strand of DNA remain intact (conserved) after replication despite their no longer being combined in the same molecule

Meselson and Stahl determined which of the three models of replication applied to E. Coli cells by...

growing E coli in one isotope of Nitrogen for several generations so that all of the amino groups of the amino acids contained this isotope, and then subjected a sample to a second isotope of nitrogen to see what percentage of the original DNA was in the subsequent replicated DNA. They did this using equilibrium density gradient centrifugation

An group of abx called 4-quinolones, commonly used to treat UTI's, kill bacteria by...

inhibiting DNA gyrase (a topoisomerase essential in bacterial DNA replication)

What is theta replication?

it is a type of semiconservative replication that takes place in circular DNA in some bacteria. It is called theta because it generates an intermediate structure that resembles the greek letter "θ". In theta replication, the double stranded DNA strand begins to unwind, producing single strands that then serve as templates. The loop that the separating strands make is called the replication bubble, and the point where unwinding is occuring is called a replication fork. If there are 2 replications forks, one on either side of the bubble, the forks proceed outward in opposite directions in a process called bidirectional replication. Both bi- and unidirectional replication produce two complete circular DNA molecules, each consisting of one old and one new strand

overall, the error rate in bacterial DNA replication is _____

less than 1 mistake per billion nucleotides

bacterial chromosomes have _____ origin(s) of replication and eukaryotic chromosomes have ____

one; many

Describe origins of replication in eukaryotes

origins of replication in different eukaryotes vary greatly in sequence, although they usually contain a number of A-T bp's. The *origin-recognition complex* (ORC) binds to the origins and initiates unwinding the DNA in those regions

Describe linear eukaryotic replication

replication begins at thousands of origins of replication along a chromosome, with replication forks spreading outward. Adjacent replicons fuse when their replication forks meet.

a segment of DNA that undergoes replication is called a ____, each of which contains a _____

replicon; origin of replication

Researchers first isolated origins of replication from ____ cells by demonstrating that certain DNA sequences confer the ability to replicate when transferred to _____. These sequences, called _____, enabled any DNA to which they were attached to replicate. They were subsequently shown to be origins of replication in _____ chromosomes

yeast plasmids (smaller circular DNA) antonomously replicating sequences (ARSs) yeast