7.2 DNA replication

replication fork/bubble

- A Y-shaped point that results when the two strands of a DNA double helix separate so that the DNA molecule can be replicated - Single fork in Prokaryotic cells - 100s of forks in Eukaryotic cells - Replication will proceed in both directions (form a bubble)

DNA replication steps

1) Helicase- unwinds the parental double helix 2) DNA topoisomerase - upstream of helices alleviating torsional strain 3) Single-strand binding proteins (SSBP) stabilize unwound DNA, aided by DNA gyrase. 4) Primase synthesizes a short RNA primer for DNA polymerase to bind to in the 5' to 3' direction to start replication on each strand. 5) DNA polymerase synthesizes the leading strand in 5' to 3' direction while the lagging strand is made discontinuously by primase making short pieces and then DNA polymerase extending these to make Okazaki fragments. 6) DNA ligase joins the Okazaki fragments together

Furthermore, all DNA polymerases only add new nucleotides onto the free 3′—OH group of a nucleotide. So how does this process ultimately begin?

1) forming of primer- To initiate DNA elongation, primase, a special RNA polymerase introduced earlier, synthesizes a short piece of single-stranded RNA that is complementary to the exposed single-stranded DNA present at the replication bubble. 2) New nucleotides-Once this primer has been formed, DNA polymerase then can add new nucleotides onto this fragment in a 5′ to 3′ fashion, using the existing DNA molecule as a template. 3) Elongation- This elongation occurs bidirectionally, and the helicase proteins described in the previous section continue to unwind the existing double-stranded DNA as the elongation events proceed. Because DNA polymerase can elongate DNA only in a 5′ to 3′ direction, only one strand of single-stranded DNA at each replication fork can be extended continuously . 4) leading strands. 5) lagging strands 6) Okazaki fragments

DNA replication: Termination

1)In E. coli, a series of ter, or termination, sites exist roughly opposite the origin of replication (oriC). 2)After bidirectional replication begins at oriC, the Tus protein binds to these ter sites and stops the progress of the replication forks. 3)Topoisomerase II then forms a transient break in the DNA, allowing the two circles to become disentangled.

Process Diagram: Role of telomerase in replication of the ends of linear chromosomes

1)On the lagging strand, a terminal RNA primer reaches the very end of the chromosome. After this terminal RNA primer is removed, a short segment of single-stranded DNA will remain. 2)To prevent the degradation of this single-stranded region, the enzyme telomerase interacts with the telomere sequence of the single-stranded DNA. A piece of RNA associated with the telomerase enzyme binds to the single-stranded DNA of the telomere. 3)Telomerase then extends the DNA, using a reverse transcriptase-like activity. 4)The enzyme subsequently moves to the newly synthesized piece of DNA and repeats the process. 5)In this manner, telomerase extends the length of the telomere. An RNA primer attaches to the newly formed piece of DNA, and DNA polymerase synthesizes new DNA, filling in the single-stranded gap.

What is the first protein to bind to the oriC region of the DNA molecule to start the replication process

1. DNA polymerase 2. Primase 3. DnaA 4. Helicase

Elongation

1. Primase synthesizes a short piece of ssRNA (in red) that is complementary to exposed ssDNA (in blue) at the replication bubble 2. DNA polymerase adds new nucleotides in the 5' to 3' direction (in green). Helicase continues to unwind in both directions from the original replication bubble creating leading and lagging strands 3. Once the DNA polymerase runs into an RNA primer a different polymerase removes the primer and adds a new nucleotide 4. Lastly DNA ligase links (or ligates the 5' phosphate and the 3' OH ends together

DNA polymerase only can add new nucleotides onto the ___________end of an existing segment of DNA. As shown in Figure 7.15, the single-stranded region at the terminus of a linear chromosome has a___________.

3′ 5′ end.

Telomerase adds complementary RNA bases to the _____________ of the DNA strand.

3′ end

Process Diagram: Initiation of DNA replication in bacteria

A)A series of proteins, including DnaA, DnaB, and DnaC, interact with oriC to help form a replication bubble. B)DnaA binds to the 9-bp repeats. Additional molecules of DnaA bind cooperatively, forming a complex with oriC, and begin denaturing the DNA. c)DnaC brings DnaB, also referred to as DNA helicase, to the complex and further unwinding of the double-stranded DNA occurs, forming a replication bubble.

Process Diagram: Initiation of DNA replication in eukarya

A)In eukarya, DNA replication initiates at several spots along the linear chromosome. B)In Saccharomyces cerevisiae, the origin recognition complex (ORC) binds to the origin, or ARS. C)Additional proteins are recruited, including Cdc6 and Cdt1. D)The minichromosomal maintenance (MCM) complex also binds and functions as the helicase. E)Additional proteins then locate to the replication bubble and begin synthesizing new DNA.

Process Diagram: DNA replication of the lagging strand in bacteria

After synthesis of an RNA primer by primase, DNA synthesis continues in a 5′ to 3′ direction with DNA pol III, adding DNA nucleotides until the newly synthesized strand of DNA encounters an RNA primer. DNA pol I then removes the RNA primer and fills in the resulting gap with deoxynucleotides. DNA ligase covalently links the DNA fragments.

Telomerase

An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells.

Bidirectional replication of DNA

As the DNA unwinds, DNA synthesis occurs in a 5′ to 3′ direction, extending from short RNA primers produced by primase. At each replication fork, one strand elongates continuously (leading strand) and the other strand elongates discontinuously (lagging strand).

Studied in S. cerevisiae ____________________has similar function to oriC

Autonomoulsly replicating sequence (ARS)

Origin of replication (oriC)

Binding site of DnaA

• Many origins of replication

Chromosomes are larger so they need multiple starting points for replication

Ligation (DNA Ligase)

Covalently links adjacent lagging strands

As the two arms of the replication fork proceed around the circular bacterial chromosome, they eventually meet on the opposite side of the circle. At this point, DNA elongation stops. In E. coli, this termination event is not random. Rather, a series of termination sequences, or ter sites, exist on the chromosome, roughly opposite of oriC.

DNA replication: Termination

The 9-bp repeats in oriC, often referred to as ____________, serve as binding sites for DnaA, a DNA-binding protein produced by the dnaA gene.

DnaA boxes

Without a functional DnaA, DNA replication does not begin, and the cell cannot grow and divide. After DnaA begins separating, or "melting," the DNA, a series of other proteins, including _____________(often referred to as helicase) and _________________(often referred to as a helicase loader), are recruited to this site to aid in the unwinding.

DnaB DnaC

Recall that in bacteria, the ____________ synthesizes short segments of RNA that bind to the single-stranded DNA. The major DNA polymerase involved in replication is the DNA polymerase III (DNA pol III) holoenzyme, a large, multisubunit complex.

DnaG primase

This enzyme adds a deoxynucleotide triphosphate to the free 3′—OH present on an existing nucleotide, removing two phosphates in the process. Elongation occurs at an impressive rate.

DnaG primase

.________________or primase, synthesizes short segments of RNA needed to prime DNA replication._____________________ attach to the newly formed single-stranded DNA to keep the strands from reannealing, and a single-stranded replication bubble forms ____________________ then begin replicating the DNA at both ends of this bubble, the replication forks.

DnaG, Single-stranded DNA-binding proteins (SSB) DNA polymerases

DNA polymerase

Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule

many origins of replication

Eukaryotes

Removal of primers DNA pol I

Excise short pieces of RNA used as primers

DNA replication: Initiation and elongation

Following the attachment of the initiator and helicase proteins to the origin of replication, the production of new DNA strands, complementary to the old strands, can occur. A series of enzymes, known as DNA polymerases, catalyze this task. However, DNA polymerases cannot simply start synthesizing a new DNA strand using a piece of single-stranded DNA as a template.

DNA replication: Origins of replication

In bacteria, DNA replication begins at a specific site on the chromosome, the origin of replication, or oriC

Replication enzyme DNA pol III

Major enzymes involved in synthesizing new DNA

• Studied in S. cerevisiae • Autonomoulsly replicating sequence (ARS) has similar function to oriC • _______________ have similar functions to DnaA-C proteins

Orc1-6 proteins

Origin recognition DnaA

Site at which replication begins. Typically AT-rich

Primase DnaG

Synthesizes short RNA oligonucleotides to which DNA polymerase adds new nucleotides

Basically, the telomere consists of a short sequence of bases repeated many times. During replication, a unique DNA polymerase, telomerase, interacts with the telomere. _________________consists of several proteins and a short piece of RNA. The RNA, it turns out, is complementary to the single-stranded DNA that forms at the end of a linear chromosome.

Telomerase

Termination of replication in circular chromosomes

Topoisomerase- an enzyme that forms a double-stranded break allowing the two circular chromosomes to separate

DnaA boxes

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

DNA polymerases require

a primer and a DNA template strand

Once the 3′ end of the lagging strand template is sufficiently elongated, DNA polymerase __________________to the ends of the chromosomes; thus, the ends of the chromosomes are replicated.

adds the complementary nucleotides

Telomerase

an enzyme in eukaryotic cells that adds a specific sequence of DNA to the telomeres of chromosomes after they divide, giving the chromosomes stability over time

semiconservative replication

because one strand of the original DNA molecule is conserved in each of the newly formed double-stranded molecules, this mechanism of replication preserves the sequence of the bases.

Cdc6, Cdt1, MCM

complex, a group of six proteins that functions as the helicase (Figure 7.10). These proteins then begin the important task of unwinding the DNA.

Topoisomerase

corrects "overwinding" ahead of replication forks by breaking, swiveling, and rejoining DNA strands

telomere

either of the repetitive nucleotide sequences at each end of a eukaryotic chromosome, which protect the chromosome from degradation

The telomerase enzyme attaches to the________________and contains a catalytic part and a built-in RNA template

end of a chromosome

ssDNA binding proteins attach and

form replication bubble

Mechanistically, the process of DNA replication in eukarya is very similar to the process of DNA replication in bacteria. Again, a primase adds short stretches of RNA to the single-stranded DNA present at the origin of replication. A DNA polymerase then begins adding nucleotides to the____________________group on the terminal nucleotide of the primer.

free 3′—OH

Elongation occurs bidirectionally in a 5′ to 3′ direction, with a __________________________present on each arm of the replication fork. Although the basic steps in the processes are similar, the specific enzymes differ (Table 7.2).

leading strand and a lagging strand

Because chromosomes in eukarya generally are___________, termination of DNA elongation presents a more challenging problem. Think about the scenario described in Figure 7.10. As _______________________ along the linear chromosome extend, the replication forks eventually will merge. Through the __________________ activity of polymerases and DNA ligase, the ________________ needed to initiate the elongation process will be removed and replaced with DNA.

linear bubbles of replication exonuclease RNA primers

Telomere

repeating nucleotide at the ends of DNA molecules that do not form genes and help prevent the loss of genes

DnaG primase

synthesizes RNA primers

DNA polymerase III

synthesizes new DNA only in the 5' to 3' direction

DNA polymerase only can add new nucleotides onto the 3′ end of an existing segment of DNA. As shown in Figure 7.15, the single-stranded region at the terminus of a linear chromosome has a 5′ end. To solve this problem, linear chromosomes of eukarya end in a special sequence known as a________________

telomere

Helicase

unwinds DNA

Helicase (DnaB)

unwinds the DNA double helix