Yeast Has Defined Origins S. cerevisiae ARS contains a conserved 11 bp ARS consensus sequence and multiple B elements ARS directs autonomous replication of plasmid DNA The ORC complex binds to the ARS during most of the cell cycle The S. pombe origin is larger and binds ORC by a distinct mechanism from Bell, Genes Dev. 16, 659 (2002)

Replication Origins in Metazoans DNA replication initiates from distinct confined sites or extended initiation zones The potential to initiate is modulated by sequence, supercoiling, transcription, or epigenetic modifications from Aladjem, Nature Rev.Genet. 8, 588 (2007) Initiation can influence initiation at an adjacent site

Some Features of Eukaryotic Replication Origins from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010) Certain characteristics are common at metazoan replication origins but are not present at all origins Different modules contribute to the selection of a given origin

Only a small subset of origins are active during a given cell cycle Constitutive origins are used all the time and are relatively rare Flexible origins are used to a different extent in different cells and follow the Jesuit Model “Many are called but few are chosen” Inactive or dormant origins are only used during replication stress or during certain cellular programs Different Classes of Replication Origins in Metazoans from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)

Chromatin Structure Influences ORC Binding from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010) Chromatin remodelling complexes can facilitate HAT binding preRC proteins can be modified by HATs

Influence of Distal Elements on Initiation from Aladjem, Nature Rev.Genet. 8, 588 (2007) Deletion of DHFR promoter allows initiation to occur within the gene Truncation of the DHFR gene confines initiation to the far end of the locus Deletion of the  -globin LCR prevents initiation within the locus Deletion of the CNS1 sequence in the Th2 cluster do not initiate within the IL13 gene

The Formation of the preRC Mcm2-7 is loaded as a double hexamer by ORC, Cdc6 and Cdt1 Sld3 and Cdc45 bind weakly to Mcm2-7 from Labib, Genes Dev. 24, 1208 (2010) Mcm2-7 helicase is inactive until S phase

Origins Are Activated at Different Times from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010) preRCs are formed during G1 on origins Heterochromatic regions replicate later than euchromatic regions

The Replicative Helicase Mcm2-7, Cdc45, and GINS (CMG complex) form the replicative helicase from Moyer et al., Proc.Nat.Acad.Sci.USA 103, (2006)

Assembly of the Replicative Helicase from Sheu and Stillman, Mol.Cell 24, 101 (2006) preRC is formed during G1 by recruitment of Mcm2-7 Phosphorylation of MCM proteins by DDK recruits GINS and stabilizes Cdc45 association

from Remus and Diffley, Curr.Opin.Cell Biol. 21, 771 (2009) Helicase Loading and Activation in DNA Replication DnaA and ORC are structural homologs Replication competence is conferred by Mcm2-7 loading and is prevented by inhibition of pre-RC proteins CDKs prevent Mcm2-7 loading and are required for helicase activation

Activation of Helicase Requires Phosphorylation of Sld2 and Sld3 G1 CDKs allow Dbf4 to accumulate DDK phosphorylates Mcm2-7 and promotes Cdc45 association CDK phosphorylates Sld2 and Sld3 and promotes association with Dpb promotes helicase activation from Botchan, Nature 445, 272 (2007)

DDK phosphorylates Mcm proteins CDK phosphorylates Sld2 and Sld3 to interact with Dpb11 GINS and Pol  are recruited to form the RPC (replisome progression complex) Activation of the helicase allows priming by Pol  Pol  extends the leading strand and Pol  extends each Okazaki fragment from Labib, Genes Dev. 24, 1208 (2010) Initiation of Chromosome Replication

Chromatin Dynamics During DNA Replication from Ransom et al., Cell 140, 183 (2010) Parental and newly synthesized H2A/H2B dimers and H3/H4 heterotetramers associate with histone chaperones Half of histones on newly replicated DNA are recycled from parental histones Nucleosome assembly is coupled to DNA replication

Replication Termination from Bell, Science 346, 418 (2014) Convergence of replication forks triggers ubiquitylation of Mcm7 Cdc48 associates with the ubiquitylated replisome and causes disassembly

from Blow and Dutta, Nature Rev.Mol.Cell Biol. 6, 476 (2005) Replication Origins are Licensed in Late M and G1 Origins are licensed by Mcm2-7 binding to form part of the pre-RC Mcm2-7 is displaced as DNA replication is initiated Licensing is turned off at late G1 by CDKs and/or geminin

from Blow and Dutta, Nature Rev.Mol.Cell Biol. 6, 476 (2005) Control of Licensing Differs in Yeasts and Metazoans CDK activity prevents licensing in yeast Geminin activation downregulates Cdt1 in metazoans

Telomeres are Specialized Structures at the Ends of Chromosomes Telomeres contain multiple copies of short repeated sequences and contain a 3’-G-rich overhang Telomeres are bound by proteins which protect the telomeric ends initiate heterochromatin formation and facilitate progression of the replication fork from Gilson and Geli, Nature Rev.Mol.Cell Biol. 8, 825 (2007)

Functions of Telomeres Telomerase is recruited by telomeric proteins and counteracts telomere shortening during DNA replication Deprotected telomeres induce checkpoint activation, the DNA damage response and DNA repair from Nandakumar and Cech, Nature Rev.Mol.Cell Biol. 14, 69 (2013)

The End Replication Problem Leading strand is synthesized to the end of the chromosome Lagging strand utilizes RNA primers which are removed The lagging strand is shortened at each cell division from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 6-49

Solutions to the End Replication Problem from de Lange, Nature Rev.Mol.Cell Biol. 5, 323 (2004) 3’-terminus is extended using the reverse transcriptase activity of telomerase Dipteran insects use retrotransposition with the 3’-end of the chromosome as a primer Kluyveromyces lactis uses a rolling circle mechanism in which the 3’-end is extended on an extrachromosomal template Telomerase-deficient yeast use a recombination- dependent replication pathway in which one telomere uses another telomere as a template Formation of T-loops using terminal repeats allow extension of invaded 3’-ends

The Action of Telomerase Solves the Replication Problem from Alberts et al., Molecular Biology of the Cell, 4 th ed. Fig 5-43 The C-strand is filled in using the extended template Telomerase-associated RNA base pairs to the G overhang Telomerase catalyzes reverse transcription to a specific site Telomerase dissociates and base pairs to a more 3’-region of the G overhang Successive reverse transcription, dissociation, and reannealing extends the G overhang

Telomeres consist of numerous short dsDNA repeats and a 3’-ssDNA overhang The G-tail is sequestered in the T-loop Shelterin is a protein complex that binds to telomeres TRF2 inhibits ATM-dependent DNA damage response Shelterin components block telomerase activity from O’Sullivan and Karlseder, Nature Rev.Mol.Cell Biol. 11, 171 (2010) Structure of Human Telomeres

POT1 binds to the 3’-ssDNA and assembles shelterin Shelterin protects the chromosome ends and inhibits the DNA damage response Telomerase is packaged in Cajal bodies and is recruited to the telomere by TPP1 during S phase Shelterin and Telomerase Recruitment from Armanios and Blackburn, Nature Rev.Genet. 13, 693 (2012)

from Bertuch and Lundblad, Curr.Opin.Cell Biol. 18, 247 (2006) Increased levels of shelterin inhibits telomerase action Telomerase Action is Restricted to a Subset of Ends Telomere length is regulated by shelterin

Maintenance of Chromosome Ends TPP1 and POT1 recruits telomerase which extends G-strands The CST complex binds to the extended G-strand and suppresses telomerase access and activity CST promotes C-strand fill-in by Pol  -Primase from Martinez and Blasco, Trends Biochem.Sci. 40, 504 (2015)

Heterochromatin at Telomeres from Martinez and Blasco, Trends Biochem.Sci. 40, 504 (2015) Telomeric heterochromatin restricts telomerase access and suppresses recombination Subtelomeric DNA is heavily methylated Chromosome ends are enriched in H3K9me3, H4K20me3 and HP1

G Overhang Generation at Telomeres TRF2 recruits Apollo at leading telomeres and initiations overhang generation POT1 binds to inhibit hyperresection Exo1 generates elongated overhangs CST is recruited by POT1 and recruits Pol  -primase to fill in C-strand from Martinez and Blasco, Trends Biochem.Sci. 40, 504 (2015)

from Marnett and Plastaras, Trends Genet. 17, 214 (2001) Endogenous DNA Damage

Biological Molecules are Labile RNA is susceptible to hydrolysis Reduction of ribose to deoxyribose gives DNA greater stability N-glycosyl bond of DNA is more labile DNA damage occurs from normal cellular operations and random interactions with the environment

Spontaneous Changes that Alter DNA Structure from Alberts et al., Molecular Biology of the Cell, 4 th ed., Fig 5-46 depurination deamination oxidation

Hydrolysis of the N-glycosyl Bond of DNA Spontaneous depurination results in loss of 10,000 bases/cell/day Causes formation of an AP site – not mutagenic from Alberts et al., Molecular Biology of the Cell, 4 th ed., Fig 5-47

Cytosine is deaminated to uracil at a rate of /cell/day Uracil is excised by uracil-DNA-glycosylase to form AP site Deamination of Cytosine to Uracil

5-Methyl Cytosine Deamination is Highly Mutagenic from Alberts et al., Molecular Biology of the Cell, 4 th ed., Fig 5-52 Deamination of 5-methyl cytosine to T occurs rapidly - base pairs with A 5-me-C is a target for spontaneous mutations

Deamination of A and G Occur Less Frequently A is deaminated to HX – base pairs with C G is deaminated to X – base pairs with C from Alberts et al., Molecular Biology of the Cell, 4 th ed., Fig 5-52