Ch. 11

DNA replication

is the process by which the genetic material is copied -The original DNA strands are used as templates for the synthesis of new strands

Okazaki fragments

Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand. Third--> Second--> First fragment (away from fork 5'->3' of fragment)

origin of replication

Site where the replication of a DNA molecule begins, consisting of a specific sequence of nucleotides. -Bacterial chromosomes have a single origin of replication thus creating a bubble

ARS elements

(for autonomously replicating sequence). ARS elements, which are about 50 bp in length, are necessary to initiate chromosome replication. ARS elements have unique features of their DNA sequences. First, they contain a higher percentage of A and T bases than the rest of the chromosomal DNA. In addition, they contain a copy of the ARS consensus sequence (ACS), ATTTAT(A or G)TTTA, along with additional elements that enhance origin function. This arrangement is similar to bacterial origins.

oriC

- origin of replication in E. coli - origin of chromosomal replication

Interpreting the Data:

-After ~ two generations, DNA is of two types: "light" and "half-heavy"This is consistent with only the semi-conservative model -After one generation, DNA is "half-heavy"This is consistent with both semi-conservative and dispersive models - each strand would contain ¼ 15N and ¾ 14N after 2 generations of dispersive

DNA polymerases I and III

involved in normal DNA replication

The effect of processivity:

-In the absence of the beta subunit -DNA pol III falls off the DNA template after a few dozen nucleotides have been polymerized -Its rate is ~ 20 nucleotides per second -In the presence of the beta subunit -DNA pol III stays on the DNA template long enough to polymerize up to 500,000 nucleotides -Its rate is ~ 750 nucleotides per second

DNA pol beta is not involved in DNA replication

-It plays a role in base-excision repair -Removal of incorrect bases from damaged DNA

lagging stand synthesis

-RNA primers must repeatedly initiate the synthesis of short segments of DNA; thus, the synthesis has to be discontinuous. The length of these fragments in bacteria is typically 1000 to 2000 nucleotides. In eukaryotes, the fragments are shorter—100 to 200 nucleotides -the synthesis of DNA also elongates in a 5ʹ to 3ʹ manner, but it does so in the direction away from the replication fork

DNA polymerases catalyzes a phosphodiester bond between the...

-innermost phosphate group of the incoming deoxynucleoside triphosphate -AND 3'-OH of the sugar of the previous deoxynucleotide -In the process, the last two phosphates of the incoming nucleotide are released -In the form of pyrophosphate (PPi)

DNA polymerase III

-responsible for synthesizing the DNA of the leading and lagging strands. This enzyme catalyzes the formation of covalent bonds between adjacent nucleotides and thereby makes the new daughter strands.

two distinct features of DNA P-3

1. DNA polymerase cannot begin DNA synthesis by linking together the first two individual nucleotides. Rather, this type of enzyme can elongate only a preexisting strand starting with an RNA primer or existing DNA strand 2.the directionality of strand synthesis. DNA polymerase can attach nucleotides only in the 5ʹ to 3ʹ direction, not in the 3ʹ to 5ʹ direction -Due to these two unusual features, the synthesis of the leading and lagging strands shows distinctive differences

There are several reasons why fidelity is high:

1. Instability of mismatched pairs 2. Configuration of the DNA polymerase active site 3. Proofreading function of DNA polymerase

When DnaA proteins are in their ATP-bound form, they bind to how many DNA boxes?

5 DnaA boxes in oriC to initiate DNA replication

AT/GC rule (Chargaff's rule)

A critical feature of the double-helix structure is that adenine hydrogen bonds with thymine, and guanine hydrogen bonds with cytosine. -the basis for the complementarity of the base sequences in double-stranded DNA. -only applies to double stranded DNA

lagging strand

A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork.

lagging strand

A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork. -multiple primers are made

pulse-chase experiment

A type of experiment in which a population of cells or molecules at a particular moment in time is marked by means of a labeled molecule (pulse) and then their fate is followed over time (chase). -They incubated E. coli cells with radiolabeled thymidine for 15 seconds and then added an excess of nonlabeled thymidine They then isolated DNA from samples of cells at timed intervals after the pulse/ chase. The DNA was denatured into single-stranded molecules, and the sizes of the radiolabeled DNA strands were determined by centrifugation. At quick time intervals, such as only a few seconds following the thymidine incubation, the fragments were found to be short, in the range of 1000 to 2000 nucleotides in length. At extended time intervals, the radiolabeled strands became much longer. At these later time points, the adjacent Okazaki fragments would have had enough time to link together

DnaC protein

Aids DnaA in the recruitment of DNA (B) helicase to the origin

Telomerase

An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells. -that prevents chromosome shortening. It recognizes the sequences at the ends of eukaryotic chromosomes and synthesizes additional repeats of telomeric sequences. -1984, Carol Greider and Elizabeth Blackburn discovered the enzyme

The enzymatic action of DNA polymerase

An incoming deoxyribonucleoside triphosphate (dNTP) is cleaved to form a nucleoside monophosphate and pyrophosphate (PPi). The energy released from this exergonic reaction allows the nucleoside monophosphate to form a covalent (ester) bond at the 3ʹ end of the growing strand. This reaction is catalyzed by DNA polymerase. PPi is released.

Why do eukaryotic chromosomes have multiple origins of replication, whereas prokaryotic chromosomes typically have only one origin?

Because eukaryotes have long, linear chromosomes, the chromosomes require multiple origins of replication so the DNA can be replicated in a reasonable length of time.

Termination Utilization Substance (TUS)

Binds to ter sequences and can stop the movement of the replication forks

Tus

Binds to ter sequences and prevents the advancement of the replication fork

DNA ligase

Covalently attaches adjacent Okazaki fragments to complete the replication process in the lagging strand -In E. coli, DNA ligase requires NAD+ to carry out this reaction, whereas the DNA ligases found in archaea and eukaryotes require ATP.

Enzymes identified in both prokaryotic and eukaryotic DNA replication?

DNA helicases, topoisomerases, single-strand binding proteins, primases, DNA polymerases, and DNA ligases

leading strand synthesis

DNA polymerase III catalyzes the attachment of nucleotides to the 3ʹ end of each primer, in a 5ʹ to 3ʹ direction. In the leading strand, one RNA primer is made at the origin, and then DNA polymerase III can attach nucleotides in a 5ʹ to 3ʹ direction as it slides toward the opening of the replication fork. -continuous

Why is DNA polymerase unable to replicate this region?

DNA polymerase synthesizes DNA only in a 5ʹ to 3ʹ direction, and it cannot link together the first two individual nucleotides; it can elongate only preexisting strands. These two features of DNA polymerase function pose a problem at the 3ʹ ends of linear chromosomes. As shown in Figure 11.25 , the 3ʹ end of a DNA strand cannot be replicated by DNA polymerase because a primer cannot be made upstream from this point. Therefore, if this problem were not solved, the chromosome would become progressively shorter with each round of DNA replication.

Based on Watson's and Crick's ideas, the hypothesis was that....

DNA replication is semiconservative -Starting material: A strain of E. coli that has been grown for many generations in the presence of 15N. All of the nitrogen in the DNA is labeled with 15N

After DNA replication is completed, one last problem may exist:

DNA replication often results in two intertwined DNA molecules known as catenanes

What prevents the DNA strands from coming back together?

DNA replication requires single-strand binding proteins that bind to the strands of parental DNA and prevent them from re-forming a double helix. In this way, the bases within the parental strands are kept in an exposed condition that enables them to hydrogen bond with individual nucleotides.

fidelity

DNA synthesis occurs with a high degree of accuracy -Mistakes during the process are extremely rare -DNA pol III makes only one mistake per 10^8bases made

What causes the DNA to bend around the complex of DnaA proteins?

DnaA proteins also bind to each other to form a complex. With the aid of other DNA-binding proteins, such as HU and IHF, results in the separation of the AT-rich region. Because only two hydrogen bonds form between AT base pairs, whereas three hydrogen bonds occur between G and C, the DNA strands are more easily separated at an AT-rich region.

template strands, or parental strands

During the replication process, the two complementary strands of DNA that come apart for the synthesis of two new strands of DNA.

Why is the fidelity so high?

First, the hydrogen bonding between G and C or A and T is much more stable than between mismatched pairs. However, this stability accounts for only part of the fidelity, because mismatching due to stability considerations accounts for 1 mistake per 1000 nucleotides. Two characteristics of DNA polymerase also contribute to the fidelity of DNA replication. First, the active site of DNA polymerase preferentially catalyzes the attachment of nucleotides when the correct bases are located in opposite strands. Helix distortions caused by mispairing usually prevent an incorrect nucleotide from properly occupying the active site of DNA polymerase. By comparison, the correct nucleotide occupies the active site with precision and undergoes induced fit, which is necessary for catalysis. The inability of incorrect nucleotides to undergo induced fit decreases the error rate to a range of 1 in 100,000 to 1 million. A second way that DNA polymerase decreases the error rate is by the enzymatic removal of mismatched nucleotides. As shown in Figure 11.16 , DNA polymerase can identify a mismatched nucleotide and remove it from the daughter strand. This occurs by exonuclease cleavage of the bonds between adjacent nucleotides at the 3ʹ end of the newly made strand.

Who recruits DNA helicase proteins to this site of separation?

Following separation of the AT-rich region, the DnaA proteins, with the help of the DnaC protein,

DNA helicase (bacteria):

In E. coli, DNA helicases bind to singlestranded DNA and travel along the DNA in a 5ʹ to 3ʹ direction to keep the replication fork moving. As shown in Figure 11.6, the action of DNA helicases promotes the movement of two replication forks outward from oriC in opposite directions. This initiates the replication of the bacterial chromosome in both directions -DnaC protein assists this process -this action generates positive supercoiling ahead of each replication fork -composed of six subunits, which form a ring around one DNA strand and migrate in the 5ʹ to 3ʹ direction.

In animals, the critical features that define origins of replication are not completely understood which means..

In many species, origins are not determined by particular DNA sequences but instead occur at specific sites along a chromosome due to chromatin structure and protein modifications.

The Fidelity of DNA Replication Is Ensured by...

Proofreading Mechanisms

Meselson and Stahl

Proved that DNA replicates in a semiconservative fashion, confirming Watson and Crick's hypothesis. Cultured bacteria in a medium containing heavy nitrogen (15N) and then a medium containing ONLY light nitrogen (14N); after extracting the DNA, they Analyze the density of the DNA by centrifugation using a CsCl gradient demonstrated that the replicated DNA consisted of one heavy strand and one light strand -They found a way to experimentally distinguish between daughter and parental strands -This experiment aims to determine which of the three models of DNA replication is correct

DNA polymerase I

Removes RNA primers, fills in gaps with DNA -composed of a single subunit. -This enzyme has a 5ʹ to 3ʹ exonuclease activity, which means that DNA polymerase I digests away the RNA primers in a 5ʹ to 3ʹ direction, leaving a vacant area. DNA polymerase I then synthesizes DNA to fill in this region. It uses the 3ʹ end of an adjacent Okazaki fragment as a primer. -DNA polymerase I would remove the RNA primer from the first Okazaki fragment and then synthesize DNA in the vacant region by attaching nucleotides to the 3ʹ end of the second Okazaki fragment. After the gap has been completely filled in, a covalent bond is still missing between the last nucleotide added by DNA polymerase I and the adjacent DNA strand that had been previously made by DNA polymerase III.

telomeres

Repeated DNA sequences at the ends of eukaryotic chromosomes. -consist of a moderately repetitive tandem array and a 3ʹ overhang region that is 12 to 16 nucleotides in length -sequence contains several guanine nucleotides and often many thymine nucleotides

DNA polymerase III

Synthesizes DNA in the leading and lagging strands -responsible for most of the DNA replication. It is a large enzyme consisting of 10 different subunits that play various roles in the DNA replication process -The α subunit actually catalyzes the bond formation between adjacent nucleotides, and the remaining nine subunits fulfill other functions

Primase

Synthesizes short RNA primers (10 to 12 nucleotides)

Existing DNA Strands Act as what???

Templates for the Synthesis of New Strands

Proofreading function of DNA polymerase

The ability to remove mismatched bases -occurs by the removal of nucleotides in the 3ʹ to 5ʹ direction at the 3ʹ exonuclease site. After the mismatched nucleotide is removed, DNA polymerase resumes DNA synthesis in the 5ʹ to 3ʹ direction.

DNA polymerase III holoenzyme

The complex of all 10 subunits together of DNA P-3

polymerase switch

The exchange of DNA pol alpha for epsilon or delta is required for elongation of the leading and lagging strands -DNA polymerase α is the only eukaryotic polymerase that associates with primase. The functional role of the DNA polymerase α/primase complex is to synthesize a short RNA-DNA primer of approximately 10 RNA nucleotides followed by 20 to 30 DNA nucleotides. This short RNA-DNA strand is then used by DNA polymerase ε or δ for the processive elongation of the leading and lagging strands, respectively. For this to happen, the DNA polymerase α/primase complex dissociates from the replication fork and is exchanged for DNA polymerase ε or δ. -occurs only after the RNA-DNA hybrid is made

leading strand

The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction. -a single primer is made at the origin of replication

the leading strand

The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction. -occurs in the direction toward the replication fork

Double helix have an antiparallel alignment which is determined by...?

The orientation of sugar molecules within the sugar-phosphate backbone. If one strand is running in the 5ʹ to 3ʹ direction, the complementary strand is running in the 3ʹ to 5ʹ direction.

Replisome

The primosome is physically associated with two DNA polymerase holoenzymes that catalyzes the synthesis of DNA

What ends replication in bacteria?

The replication forks eventually meet at the opposite side of the bacterial chromosome

How does telomerase function?

The telomerase enzyme contains both protein subunits and RNA. The RNA part of telomerase contains a sequence complementary to the DNA sequence found in the telomeric repeat. This allows telomerase to bind to the 3ʹ overhang region of the telomere. Following binding, the RNA sequence beyond the binding site functions as a template allowing the synthesis of a six-nucleotide sequence at the end of the DNA strand. This is called polymerization, because it is analogous to the function of DNA polymerase. Following polymerization, the telomerase can then move—a process called translocation—to the new end of this DNA strand and attach another six nucleotides to the end. This binding-polymerization-translocation cycle occurs many times in a row, thereby greatly lengthening the 3ʹ end of the DNA strand in the telomeric region. The complementary strand is then synthesized by primase, DNA polymerase, and DNA ligase, as described earlier in this chapter.

semiconservative model

Type of DNA replication in which the replicated double helix consists of one old strand, derived from the old molecule, and one newly made strand. -The double-stranded DNA contains one parental and one daughter strand following replication

Bacterial Chromosomes Contain _____________ Origin of Replication

a Single

origin recognition complex (ORC)

a complex of six proteins found in eukaryotes that is necessary to initiate DNA replication -originally identified in yeast as a protein complex that binds directly to ARS elements. DNA replication at the origin begins with the binding of ORC, which usually occurs during G1 phase. Other proteins of the preRC then bind, including MCM helicase

MCM helicase

a group of eukaryotic proteins needed to complete a process called DNA replication licensing, which is necessary for the formation of two replication forks at an origin of replication

DnaA proteins

a protein that binds to the dnaA box sequence at the origin of replication in bacteria and initiates DNA replication -This binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex

To prevent the loss of genetic information due to chromosome shortening,.....

additional DNA sequences are attached to the ends of telomeres

Eukaryotic DNA polymerases

alpha - lagging, beta = Base Excision repair, delta = leaDing strand, gaMMA = Momma = mitochondrial epsilon=leading strand synthesis -DNA polymerase γ functions in the mitochondria to replicate mitochondrial DNA, whereas α, ε, and δ are involved with DNA replication in the cell nucleus during S phase.

Subunit Composition of DNA Polymerase III Holoenzyme

alpha, epsilon, beta

β subunit of the holoenzyme

also known as the clamp protein, promotes the association of the holoenzyme with the DNA as it glides along the template strand (refer back to Table 11.2). The β subunit forms a dimer in the shape of a ring; the hole of the ring is large enough to accommodate a double-stranded DNA molecule, and its width is about one turn of DNA. A complex of several subunits functions as a clamp loader that allows the DNA polymerase holoenzyme to initially clamp onto the DNA

Three types of DNA sequences are found within oriC:

an AT-rich region, DnaA box sequences, and GATC methylation sites.

exonuclease

an enzyme that removes successive nucleotides from the end of a polynucleotide molecule

S phase replication

at the beginning of S phaseChromosome replicationBegins at the multipleOrigins of replicationReplication forks move BidrectionallyAt the end of S phase, Replication forks have merged

Lesion-replicating polymerases

attracted to the damaged DNA and have special properties that enable them to synthesize a complementary strand over the abnormal region. Each type of lesion-replicating polymerase may be able to replicate over a different kind of DNA damage.

Is bacterial or eukaryotic DNA replication better understood?

bacterial -experiments done on yeast and mammals to better understand

Another key difference between bacterial and eukaryotic DNA replication is the way that RNA primers are removed.

bacterial RNA primers are removed by DNA polymerase I. By comparison, a DNA polymerase enzyme does not play this role in eukaryotes. Instead, an enzyme called flap endonuclease is primarily responsible for RNA primer removal.

DNA replication proceeds ________ from many origins of replication during S phase of the cell cycle.

bidirectionally -The multiple replication forks eventually make contact with each other to complete the replication process.

single-strand binding proteins

bind to the unpaired DNA strands, keeping them from re-pairing

How is replication initiated?

by the Binding of DnaA Protein to the Origin of Replication

DNA polymerase

catalyze the covalent attachment between the phosphate in one nucleotide and the sugar in the previous nucleotide. The formation of this covalent (ester) bond requires an input of energy. Prior to bond formation, the nucleotide about to be attached to the growing strand is a dNTP. It contains three phosphate groups attached at the 5ʹ-carbon atom of deoxyribose. The dNTP first enters the catalytic site of DNA polymerase and binds to the template strand according to the AT/GC rule. Next, the 3ʹ-OH group on the previous nucleotide reacts with the phosphate group adjacent to the sugar on the incoming nucleotide. The breakage of a covalent bond between two phosphates in a dNTP is a highly exergonic reaction that provides the energy to form a covalent (ester) bond between the sugar at the 3ʹ end of the DNA strand and the phosphate of the incoming nucleotide. The formation of this covalent bond causes the newly made strand to grow in the 5ʹ to 3ʹ direction, pyrophosphate (PPi) is released.

DNA polymerase slides along the template strand as it synthesizes a new strand by connecting_________ in a 5ʹ to 3ʹ direction.

deoxyribonucleoside triphosphates (dNTPs)

processive enzyme

describes the behavior of DNA polymerase III, which can perform thousands of rounds of catalysis without dissociating from its substrate (the template DNA strand) -This means it does not dissociate from the growing strand after it has catalyzed the covalent joining of two nucleotides. Rather, it remains clamped to the DNA template strand and slides along the template as it catalyzes the synthesis of the daughter strand -(allowing DNA synthesis to occur at 750 nucleotides/second)

positive supercoiling

double helix is overwound

dispersive model

each strand of both daughter molecules contains a mixture of old and newly synthesized DNA -Parental and daughter DNA are interspersed in both strands following replication

Dna2 nuclease/helicase

enzyme can cut a long flap, thereby generating a short flap. The short flap is then removed via flap endonuclease.

How many DNA polymerases does E. coli have?

five -designated polymerase I, II, III, IV, and V

catenanes

form when the two circular daughter chromosomes do not separate -Fortunately, catenanes are only transient structures in DNA replication. In E. coli, topoisomerase II introduces a temporary break into the DNA strands and then rejoins them after the strands have become unlocked. This allows the catenanes to be separated into individual circular molecules.

Feature of eukaryotic cells..

have larger, linear chromosomes, the chromatin is tightly packed within nucleosomes, and cell cycle regulation is much more complicated

The catalytic subunit of all DNA polymerases has a structure that resembles a......?

human hand -the template DNA is threaded through the palm of the hand; the thumb and fingers are wrapped around the DNA. The incoming deoxyribonuleoside triphosphates (dNTPs) enter the catalytic site, bind to the template strand according to the AT/GC rule, and then are covalently attached to the 3ʹ end of the growing strand. DNA polymerase also contains a 3ʹ exonuclease site that removes mismatched bases, as described later.

prereplication complex (preRC)

in eukaryotes, an assembly of at least 14 different proteins, including a group of 6 proteins called the origin recognition complex (ORC), that acts as the initiator of eukaryotic DNA replication.

DNA replication licensing

in eukaryotes, occurs when MCM helicase is bound at an origin, enabling the formation of two replication forks -can initiate DNA synthesis. During S phase, DNA synthesis begins when preRCs are acted on by at least 22 additional proteins that activate MCM helicase and assemble two divergent replication forks at each replication origin

What happens after the double helix has separated?

individual nucleotides have access to the template strands. Hydrogen bonding between individual nucleotides and the template strands must obey the AT/GC rule. To complete the replication process, a covalent bond is formed between the phosphate of one nucleotide and the sugar of the previous nucleotide.

dimeric DNA polymerase

is used to describe two DNA polymerase holoenzymes that move as a unit toward the replication fork. For this to occur, the lagging strand is looped out with respect to the DNA polymerase that synthesizes the lagging strand. This loop allows the lagging-strand polymerase to make DNA in a 5ʹ to 3ʹ direction yet move toward the opening of the replication fork. Interestingly, when this DNA polymerase reaches the end of an Okazaki fragment, it must be released from the template DNA and "hop" to the RNA primer that is closest to the fork. The clamp loader complex (see Table 11.2), which is part of DNA polymerase holoenzyme, then reloads the enzyme at the site where the next RNA primer has been made. Similarly, after primase synthesizes an RNA primer in the 5ʹ to 3ʹ direction, it must hop over the primer and synthesize the next primer closer to the replication fork

Flap endonuclease

it removes small pieces of RNA flaps that are generated by the action of DNA polymerase δ. -cannot remove long flaps -DNA polymerase δ elongates the left Okazaki fragment until it runs into the RNA primer of the adjacent Okazaki fragment on the right. This causes a portion of the RNA primer to form a short flap, which is removed by the endonuclease function of flap endonuclease. As DNA polymerase δ continues to elongate the DNA, short flaps continue to be generated, which are sequentially removed by flap endonuclease. Eventually, all of the RNA primer is

termination (ter) sequences

located On the opposite side of the E. coli chromosome from oriC in a pair -one of the ter sequences designated T1 prevents the advancement of the fork moving left to right, but allows the movement of the other fork. Alternatively, T2 prevents the advancement of the fork moving right to left, but allows the advancement of the other fork. In any given cell, only one ter sequence is required to stop the advancement of one replication fork, and then the other fork ends its synthesis of DNA when it reaches the halted replication fork. In other words, DNA replication ends when oppositely advancing forks meet, usually at T1 or T2. Finally, DNA ligase covalently links the two daughter strands, creating two circular, double-stranded molecules.

n 1968, Huberman and Riggs provided evidence for the ..

multiple origins of replication

DNA polymerases II, IV, and V

play a role in DNA repair and the replication of damaged DNA

replication forks

portions of DNA where the double helix separates during DNA replication -site where the parental DNA strands have separated and new daughter strands are being made. Eventually, these replication forks meet each other on the opposite side of the bacterial chromosome to complete the replication process. -2 forks

DNA replication in begins with assembly of the...

prereplication complex (preRC)

Each fragment contains a short RNA primer at the 5ʹ end, which is made by a??

primase -The remainder of the fragment is a strand of DNA made by DNA polymerase III.

DNA helicase (DnaB)

prokaryotes; opens helix and binds primase to form primosome. -breaks the hydrogen bonds between the two strands, thereby generating two single strands. Two DNA helicases begin strand separation within the oriC region and continue to separate the DNA strands beyond the origin. These proteins use the energy from ATP hydrolysis to catalyze the separation of the doublestranded parental DNA.

To complete the synthesis of Okazaki fragments within the lagging strand, three additional events must occur:

removal of the RNA primers, synthesis of DNA in the area where the primers have been removed (DNA P-1), and the covalent attachment of adjacent fragments of DNA (DNA ligase)

To act as a template for DNA replication, the strands of a double helix must.....?

separate

DnaA box sequences

serves as a recognition site for the binding of the DnaA protein, which is involved in the initiation of bacterial DNA replication

What allows DNA polymerase III to begin the synthesis of complementary daughter strands of DNA?

synthesis of RNA primers by primase

RNA primers (rather than DNA)

synthesized by the linkage of ribonucleotides via an enzyme known as primase. This enzyme synthesizes short strands of RNA, typically 10 to 12 nucleotides in length. -short strands of RNA that start, or prime, the process of DNA replication

The Ends of Eukaryotic Chromosomes Are Replicated by

telomerase

Replication Is Terminated When....

the Replication Forks Meet at the Termination Sequences

telomerase reverse transcriptase (TERT)

the enzyme within telomerase that uses RNA as a template to make DNA -two identical subunits

bidirectionally

the phenomenon in which two replication forks move in opposite directions, outward from the origin of the bacterial chromosome -synthesis of new daughter strands is initiated

phosphodiester bond

the type of bond that links the nucleotides in DNA or RNA. joins the phosphate group of one nucleotide to the hydroxyl group on the sugar of another nucleotide -involves two ester bonds. In comparison, as a DNA strand grows, a single covalent (ester) bond is formed between adjacent nucleotides. The other ester bond in the phosphodiester linkage—the bond between the 5ʹ-oxygen and phosphorus—is already present in the incoming nucleotide

At the molecular level, eukaryotic origins of replication have been extensively studied in what organism?

the yeast Saccharomyces cerevisiae -In this organism, several replication origins have been identified and sequenced.

In S. cerevisiae, origins of replication are determined primarily by..

their DNA sequences

What happens after the two parental DNA strands have been separated and the supercoiling relaxed (positive supercoiling)?

they must be kept that way until the complementary daughter strands have been made

Primosome

tracks along the DNA, separating the parental strands and synthesizing RNA primers at regular intervals along the lagging strand. By acting within a complex, the actions of DNA helicase and primase can be better coordinated helicase + primase -associates with two DNA polymerase enzymes to form a replisome -complex leads the way at the replication fork

Topoisomerase type II (DNA gyrase)

travels in front of DNA helicase and alleviates positive supercoiling

daughter strands

two newly made strands -DNA is replicated so that both copies retain the same information—the same base sequence—as the original molecule.

conservative model

two parental strands reassociate after acting as templates for new strands thus restoring the parental double helix -Both parental (template) strands stay together after DNA replication

One reason why telomeric repeat sequences are needed is because DNA polymerase is....

unable to replicate the 3ʹ ends of DNA strands

clamp-loader protein

y subunit is needed for beta to initially clamp onto the DNA

Functions of key proteins involved with DNA replication in bacteria:

• DNA helicase (DNAB) breaks the hydrogen bonds between the DNA strands. • Topoisomerase II (DNA gyrase) alleviates positive supercoiling. • Single-strand binding proteins keep the parental strands apart. • Primase synthesizes an RNA primer (10-12 nucleotides). • DNA polymerase III synthesizes a daughter strand of DNA. • DNA polymerase I excises the RNA primers and fills in with DNA. • DNA ligase covalently links the Okazaki fragments together