Lecture 6: DNA Replication II

DNA Replication Side notes: Leading and lagging strand synthesis is coordinated (bacteria) Leading and lagging strand synthesis is coupled at the replication fork DNA on the lagging strand is looped The DNA loop of the lagging strand needs to be released from polymerase frequently (every couple of seconds) - a new Okazaki fragment is produced every 1-2 kb In bacteria, the replication proteins are associated in the replication complex, or replisome The bacterial replisome has two copies of DNA polymerase III, sliding clamps and clamp loader The polymerases are linked by tau, which associates with the clamp loader and helicase Leading and lagging strand synthesis is coordinated (eukaryotes) Leading and lagging strand synthesis in eukaryotes is coordinated at the replication fork by Ctf4 DNA polymerase epsilon (leading strand) and delta (lagging strand) are present at the replication fork Ctf4 couples the CMG helicase, polymerase epsilon, and the polymerase alphaprimase at the fork There is no eukaryotic equivalent of bacterial tau, so polymerase epsilon and delta are not linked - instead, strands are coupled through the Ctf4-polymerase alpha-primase hub Okazaki fragment maturation in bacteria DNA is synthesized on both the leading and lagging strands at the replication fork Because DNA polymerase synthesizes DNA in a 5' to 3' direction, DNA is made In short discontinuous fragments on the lagging strand - Okazaki fragments Okazaki fragments are joined after synthesis - Okazaki fragment maturation this is different in eukaryotes and bacteria In E. coli, DNA polymerase III disassociates from the DNA when it reaches the next RNA primer, but the sliding clamp remains attached to the DNA DNA polymerase I removes the RNA primer and then fills in the gap with DNA, but cannot attach the end of the synthesized DNA to the next DNA fragment, leaving a "nick" in the DNA DNA lipase seals the DNA "nick" Okazaki fragment maturation in eukaryotes Removal of the RNA primer and filling in with new DNA is different in eukaryotes DNA polymerase delta or epsilon continues to make DNA when it meets the RNA primer This displaces the existing RNA primer and DNA from the template strand, making a flap Flap endonuclease (Fen 1) cleaves the flap DNA A homolog of Fen 1 is required in archaea, suggesting this mechanism is conserved in archaea and eukaryotes, but not bacteria DNA lipase seals the DNA "nick" Termination of replication has 3 steps: Resolving torsional stress when two replication forks approach one another Meeting of replication forks and generation of complete DNA strands Dissociation of the replisome from DNA DNA Replication Side notes: Topoisomerases relax DNA supercoiling As DNA is unwound by helicases, torsional stress is introduced into the DNA strand separation at the fork results in overwinding ahead of the fork - positive supercoiling DNA ahead of the fork gains one positive supercoil for each DNA turn that is unwound The increased supercoiling ahead of the fork makes strand separation more difficult, especially if too replisomes are approaching one another Topoisomerases resolve the overwinding by transiently breaking DNA and allowing supercoils to relax Topoisomerases also unlink replication products Replication of bacterial circular chromosomes results in daughter molecules that are still interlinked - ‘catenated' Circular chromosomes are unlinked - ‘decatenated' - by type 1A topoisomerase at step 3 or type II topoisomerases at step 4 Types of topoisomerases Type I topoisomerases cleave one strand of the DNA duplex - they have an active tyrosine -OH that attacks the phosphodiester of one strand (no ATP required) Type 1A topoisomerases pass the other strand through the break Type 1B topoisomerases allow the free end to swivel to release supercoils before relegation Type II topoisomerases cleave double-stranded DNA and pass another duplex through the break (ATP required) Type 1A and Type II topoisomerases act by strand passage Type 1A and type II topoisomerases act by strand passage - a single strand for type 1A and double-stranded DNA for type II Type II topoisomerases are dimeric enzymes that cleave both strands of the DNA, with each subunit forming a covalent bond between active site tyrosine and the 5' end of the cleaved strand Conformational changes allow another double-stranded segment of DNA to pass through the gap made in the DNA - this step requires ATP hydrolysis Termination occurs at specific sites in bacterial chromosomes Replication forks meet in a termination zone that has 10 ter sites Ter sites bound by the Tus protein are orientation specific and allow the fork to pass only in one direction - trapping the replication forks in the termination zone The replisomes of opposing forks cause flap displacement when they pass each other - this is filled by a mechanism similar to Okazaki fragment maturation (DNA pol I) Termination occurs at multiple sites in eukaryotic chromosomes Replication forks meet at many different sites due to multiple replication origins When replisomes meet, the CMG complexes move past each other on the leading strand As the replisomes move past each other, a flap of newly synthesized DNA is displaced The flaps are resolved by the same machinery that processes Okazaki fragments This leaves the double-stranded DNA entwined, and this is resolved by DNA Replication Side notes: Topoisomerase II Regulation of initiation in bacteria Initiation of replication in bacteria is tightly regulated by factors such as cell size and nutritional state DNAA binds and multimerizes at oriC when bound to ATP After binding, DNAA unwinds DNA for replication initiation After initiation, another protein, Hda, stimulates hydrolysis of the DNAA-ATP to ADP DNAA-ADP dissociates and cannot reinitiate origin firing, preventing overstimulation of replication initiation This regulatory inactivation of DNAA (RIDA) mechanism blocks replication initiation RIDA is the major regulatory step preventing initiation DNAA binding concentration is also effectively lowered by newly copied origin Initiation at oriC can also be prevented by methylation DNA adenine methylase (Dam methylase) methylates the A residue in the sequence GATC 11 GATC sites in oriC overlap the DNAA binding sites Just after replication, only one strand of DNA is methylated (hemi-methylation) SeqA binds to these hemi-methylated GATC sites and blocks binding of methylase. This inhibits methylation and blocks access by DNAA This is only a temporary block - the origin is usually fully methylated ten minutes after initiation Regulation of initiation in eukaryotes Eukaryotes have multiple replication origins, but they must only fire a maximum of once per cell cycle Origins are selected in G1 (‘origin licensing') Origins are prepared for activation in G1 by loading of the origin recognition complex (orc) and other factors, including helicase Helicases are loaded in a two-step process facilitated by Cdt1 and Cdc6, but the helicase complexes are inactive In S phase, helicases activate to inmate origin unwinding - once fired, origins cannot be reused until the next G1 Cell cycle kinases are involved in restricting initiation steps and origin firing to the appropriate timing Mechanisms to prevent helicase reloading outside of G1 Yeast does not break down its nucleus in mitosis, and regulation is achieved by exporting proteins from the nucleus In metazoa, the main mechanism is proteolysis of replication complex proteins after S-phase Geminin (unique to metazoa) is an additional control layer - this binds to Cdt1 and prevents it loading MCM to the origin during S, G2, and M phases Replication was problems at the ends of chromosomes The mechanism for lagging strand synthesis cannot replicate the very end of a linear chromosome - called the end-replication problem As a consequence, substantial portions of chromosome ends could be lost over several rounds of replication One cause of this is RNA primer removal, which can leave a gap that cannot be filled in Side notes: Another problem is dissolution of the replication fork when the leading strand is finished, before lagging strand synthesis is complete This can lead to the loss of the terminal Okazaki fragment Chromosome ends = telomeric repeats Many eukaryotic chromosome ends have simple sequence repeats - telomeres Telomeres range in length from -100 bp to > 20,000 bp, depending on species The ends of the telomeres get shorter after each round of replication Telomeres can be elongated by adding new specific telomere sequences at the ends Telomere-binding proteins protect chromosome ends There are three classes of telomere binding proteins: End-binding proteins protect the G-rich overhang Double-stranded binding proteins bind along the length Proteins that associate with the DNA binding proteins In humans, the complex of protective proteins is called Shelterin Telomere-binding proteins regulate length - mutational changes or overexpression of Shelterin results in telomere shortening or elongation Loss of telomere function causes problems for the cell. The unprotected end is "repaired" as a DNA break, which causes broken and aberrantly joined chromosomes Telomerase maintains telomere length Telomerase is a unique polymerase made of a catalytic protein reverse transcriptase and an integral bound RNA molecule The catalytic protein is called telomerase reverse transcriptase (TERT), and is well conserved in eukaryotes The RNA provides the template for synthesis of telomere repeats, and has a role in enzyme activity - the sequence is not conserved among organisms, but secondary structure is conserved First, telomerase binds to single-stranded telomere DNA and the 3' end of the DNA base-pairs with the telomerase RNA The enzyme uses the RNA as a template to synthesize DNA telomeric sequence Once the end of the template is reached, telomerase realigns with the new DNA end and the cycle is repeated Telomere length in most eukaryotes is maintained by telomerase Telomerase adds telomere DNA sequences into the chromosome ends Telomerase synthesizes one strand of the telomere - the G-rich strand - at the 3' end The complementary C-rich strand is filled in by DNA polymerase alpha The action of telomerase results in net elongation of the telomere sequence, which counterbalances loss due to incomplete end-replication It is not yet understood how the distribution of telomere length in a cell is regulated, but telomere binding proteins are thought to be involved Two factors must help establish the equilibrium: Number of telomeres elongated in a cell cycle How many repeats are added in a single binding event Mutations in telomere binding proteins can lead to aberrant telomere lengthening The DNA damage response is induced by short telomeres - loss of telomerase or other factors lead to progressive telomere shortening Shortening can be tolerated somewhat, but when telomeres become critically Side notes: Short, they no longer protect chromosome ends, triggering cell cycle arrest and/or cell death Maintenance of telomere length is important - mutations in telomerase cause inherited degenerative diseases, known as telomere syndromes Telomerase is also important in cancer: > 90% of human tumors have active telomerase that maintains their telomeres, reducing the growth barrier and allowing indefinite growth - these cells are termed ‘immortalized’ How does replication proceed through chromatin? Histones must be recruited to the correct places in the daughter DNA molecules after replication DNA modifications like methylation must also be kept The correct recruitment of histones, and maintenance of chemical modifications such as methylation underpin epigenetic inheritance Eukaryotic DNA is packaged in nucleosomes - these are disrupted by the replication fork, then re-bind after the fork has passed Half of the parental H3 and H4 histones go to one daughter, half to the other this is parental histone segregation, and it allows the histone modification state to be inherited by both cells New H3 and H4 histones are deposited and they are modified in line with the old histones CMG helicase at the replication fork disrupts nucleosomes in its path The histone chaperone FACT, along with ASF protein, helps to disrupt the nucleosome ahead of the fork and reassemble the old H3/H4 behind the fork FACT is also involved in deposition of new nucleosomes behind the fork New H2A and H2B histones are loaded to make a half nucleosome, and new H3/H4 histones are assembled together by CAF1 protein FACT then interacts with the new H3/H4 histones and CMG helicase and deposits them with the H2A/H2B half nucleosome to make a complete new nucleosome Synthesis of histones is tightly coupled to DNA synthesis Newly-synthesized H3 and H4 are acetylated at specific lysines in their Nterminal tails Deposition of new acetylated H3/H4 histones on newly replicated DNA is performed by chromatin assembly factor 1 (CAF1) CAF1 binds strongly to H3/H4 and to the sliding clamp to target H3/H4 to DNA behind the fork ASF1 binds a half nucleosome tetramer (2 x H3 and 2 x H4) and, along with CAF1, deposits this on DNA Deposition of a H2A/H2B tetramer then follows Once deposited, new histones are deacetylated ready for modification in line with existing 'Old' histones