03 Eukaryotic DNA Replication

Eukaryotes Use Several DNA Polymerases

*Roman numeral = Prokaryotes* *Alpha Delta = Eukaryotes* -More complex in terms of the amount of DNA to be replicated and the number of proteins required (estimated at >27 in yeast and mammals) compared to prok -Animal cells contain at least 13 distinct DNA polymerases, named with *Greek letters* according to their order of discovery. -A newer classification scheme uses sequence homology to group eukaryotic as well as prokaryotic polymerases into six families: A, B, C, D, X, and Y -Three main enzymes involved in replicating eukaryotic nuclear DNA: *polymerases α, δ, and ε,* which are all members of the B-family of polymerases (Table 25-2).

DNA polymerase δ (pol δ)

-*5' → 3' direction* under the direction of a ssDNA template. -does not associate with a primase; contains a 3' → 5' exonuclease active site.; processivity is essentially unlimited -Requires association with proliferating cell nuclear antigen (PCNA). -forms a trimeric ring with almost identical structure (and presumably function) as the E. coli β2 sliding clamp -PCNA and the β clamp exhibit no significant sequence identity, even when their structurally similar portions are aligned. -During replication, RFC (replication factor C, a clamp loader that is the eukaryotic counterpart of the E. coli γ complex) loads PCNA onto the template strand near the primer. -This *displaces pol α*, allowing pol δ to bind and *processively* extend the new DNA strand alpha, delta= lagging episilon= leading

Telomeres Form G-Quartets

-*Guanine-rich polynucleotides* are notoriously difficult to work with. This is because of their propensity to aggregate via Hoogsteen-type base pairing to form cyclic tetramers known as *G-quartets* -The* G-rich overhanging strands of telomeres* fold back on themselves to form a hairpin, two of which associate in an antiparallel fashion to form stable complexes of stacked G-quartets -Such structures presumably serve as binding sites for capping proteins, which may *help regulate telomere length* and prevent activation of DNA repair mechanisms that recognize the ends of broken DNA molecules.

Nucleosomes Reassemble behind the Replication Forks

-*Nucleosomes just ahead of the replication fork disassemble and the freed histones*, either individually or as dimers or tetramers, immediately reassociate with the emerging daughter duplexes. -*DNA replication* (which occurs in the nucleus) is *coordinated* with histone protein synthesis in the cytosol so that new histones are available in the required amounts

DNA polymerase α (pol α)

-5' → 3' direction under the direction of a ssDNA template. -*no exonuclease activity and therefore cannot proofread* -moderately processive (polymerizing ~100 nucleotides at a time) -associates tightly with a primase, indicating that it is involved in initiating DNA replication. -The pol α/primase complex synthesizes a 7- to 10-nt RNA primer and extends it by an additional 15 or so deoxynucleotides. -lack of proofreading activity is not problematic, since the first few residues of newly synthesized DNA are typically removed and replaced along with the RNA primer.

Telomerase Extends Chromosome Ends

-DNA polymerase cannot synthesize the extreme 5' end of the lagging strand -Linear chromosomes would be shortened at both ends by at least the length of an RNA primer with each round of replication. -gradual truncation of chromosomes with each round of DNA replication contributes to the normal senescence of cells. -cells in culture can undergo only a limited number of doublings (20-60) before they reach senescence (a stage in which they cease dividing) and eventually die.

DNA polymerase γ (pol γ)

-DNA polymerase γ (pol γ), an A-family enzyme, occurs exclusively in the mitochondrion, where it presumably replicates the mitochondrial genome. -Chloroplasts contain a similar enzyme.

DNA polymerase ε (pol ε)

-DNA polymerase ε (pol ε) superficially resembles pol δ but is highly processive in the absence of PCNA. -essential for DNA replication -pol ε has been proposed to function as the leading-strand polymerase while pol α and pol δ cooperate to synthesize the lagging strand (pol δ also participates in leading strand synthesis as well)

Additional Enzymes Participate in Eukaryotic DNA Replication

-Helicase: required to pry apart the two template strands In eukaryotes, this function belongs to the heterohexameric complex known as *MCM*. -*Single-stranded DNA* becomes coated with the trimeric *RPA*, the *eukaryotic equivalent of the bacterial SSB*. -The eukaryotic replisome also includes additional proteins that have no prokaryotic counterparts and whose functions are not yet understood. -*Euks lack a nick-translating polymerase like E. coli Pol I*. -The *RNA primers of Okazaki fragments are removed through the actions of two enzymes*: -*RNase H1 removes most of the RNA* -Leaves only a 5'-ribonucleotide adjacent to the DNA, which is then removed through the action of flap endonuclease-1 (FEN1). -*FEN1*: proofreading function -endonuclease that *excises mismatch-containing oligonucleotides* up to 15 nt long from the 5' end of an annealed DNA strand. -The excised segment is later replaced by *pol δ and DNA ligase seals the last nick*.

Eukaryotic DNA Is Replicated from Multiple Origins

-Replication fork movement in eukaryotes is *~10 times slower* than in prokaryotes. Since a eukaryotic chromosome typically contains *60 times more DNA than does a prokaryotic chromosome*, its bidirectional replication from a single origin, as in prokaryotes, would require ~1 month. -eukaryotic chromosomes contain multiple origins, one every 3 to 300 kb, depending on both the species and the tissue, so replication requires a few hours to complete. -In yeast, the *initiation of DNA replication occurs at autonomously replicating sequences (ARS)* conserved 11-bp sequences adjacent to easily unwound DNA. -In mammalian genomes, replication origins: do not exhibit sequence conservation; all support the binding of a six-subunit origin recognition complex (ORC). *Large chromosomes!* -pre-replication complex (pre-RC) is the combination of binding of ORC and helicase MCM -Additional ATP-hydrolyzing proteins help assemble the helicase MCM. -pre-RC is not competent to initiate replication until it has been activated by other factors that control cell cycle progression (G1, S, G2, M) -separate control of pre-RC assembly and activation allows cells to select replication origins before MCM unwinds the template DNA and replication commences. -Once DNA synthesis is under way, no new pre-RC complexes can form at that origin -DNA is replicated once and only once per cell cycle. -Initiation sites are uniformly distributed across the genome in early embryogenesis, when cell division is rapid. -After cells have differentiated, the distribution of replication origins changes, possibly reflecting patterns of gene expression and/or alterations in DNA packaging in different cell types. -clusters of 20 to 80 adjacent replicons (replicating units; DNA segments that are each served by a replication origin) are activated simultaneously. chromosomal regions are not all replicated simultaneously. -New sets of replicons are activated until the entire chromosome has been replicated. -DNA replication proceeds in each direction from the origin of replication until each replication fork collides with a fork from the adjacent replicon. -Eukaryotes appear to lack termination sequences analogous to the Ter sites in E. coli. *No termination sequences*

Reverse Transcriptase

-Reverse transcriptase (RT) is an *essential enzyme of retroviruses*, which are RNA-containing eukaryotic viruses human immunodeficiency virus (HIV, the causative agent of AIDS). -RT, which was independently discovered in 1970 by Howard Temin and David Baltimore, *synthesizes DNA in the 5' → 3' direction from an RNA template*. -*RT catalyzes the first step in the conversion of the virus' single-stranded RNA genome to a double-stranded DNA*. -Reverse transcriptase *lacks a proofreading exonuclease* function and is highly error-prone. -RT: useful tool in genetic engineering; transcribe mRNAs to complementary strands of DNA (cDNA).

Telomerase, Aging, and Cancer

-Telomeres can protect against the loss of essential genes near the ends of chromosomes -Also telomeres provide a protective process called capping -*Capping helps to prevent the end to end fusion of chromosomes* (two chromosome ends together), a process that could result in chromosomal instability and chromosomal breakage during mitosis -Proteins specifically bind to the telomeric DNA and is mediated telomere length -There is a *strong correlation between the initial telomere length in a cell and its proliferative capacity*. -*Cells* that initially have relatively *short telomeres undergo significantly fewer doublings* than cells with longer telomeres. -Fibroblasts from individuals with progeria (a rare disease characterized by rapid and premature aging resulting in childhood death) have short telomeres, an observation that is consistent with their known reduced proliferative capacity in culture. -In contrast, *sperm (which are essentially immortal)* have telomeres that do not vary in length with donor age, which indicates that telomerase is active during germ-cell growth.

Telomeres Are Built from an RNA Template

-Telomeres: ends of eukaryotic chromosomes (Greek: telos, end) -*Consists of 1000 or more tandem repeats of a short G-rich sequence* (TTGGGG in the protozoan Tetrahymena and TTAGGG in humans) *on the 3'-ending strand* of each chromosome end -*Telomerase*: *ribonucleoprotein* (a complex consisting of protein and RNA) synthesized and maintained telomeric DNA -Telomoerase uses its RNA template (451 nt in humans) that is complementary to the repeating telomeric sequence and adds to the 3' end of the DNA. -Telomerase functions similarly to reverse transcriptase; its protein component is homologous to reverse transcriptase. -*Telomerase repeatedly translocates to the new 3' end of the DNA strand, adding multiple telomeric sequences to the DNA* -*Lagging-strand synthesis uses extended 3' end to replicate telomeric DNA* -*3' end is G-rich: 100-300 nucleotide overhang remains*

Reverse Transcriptase, Virus

-Virus enters a cell, -Its RT uses the viral *RNA as a template* to synthesize a complementary DNA strand, yielding an RNA-DNA hybrid helix. The DNA synthesis is primed by a host cell tRNA whose 3′ end unfolds to base-pair with a complementary segment of viral RNA. -The viral RNA strand is then nucleolytically degraded by an RNase H (an RNase activity that hydrolyzes the RNA of an RNA-DNA hybrid helix). -Finally, the *DNA strand acts as a template for the synthesis* of its complementary DNA, yielding dsDNA that is then integrated into a host cell chromosome.