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The initiation of yeast DNA replication Questions? Contact Van Andel Research Institute Grand Rapids,

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Presentation on theme: "The initiation of yeast DNA replication Questions? Contact Van Andel Research Institute Grand Rapids,"— Presentation transcript:

1 The initiation of yeast DNA replication Questions? Contact Van Andel Research Institute Grand Rapids, MI BMB801 Lecture Dr. Michael Weinreich 10/4/06

2 Initiation of DNA Replication Essential for cell growth, development, integrity of the genetic information Each origin initiates replication only once per cell cycle Highly regulated. Commitment to DNA replication is an irreversible decision Many initiation proteins are upregulated in cancer cells

3 Initiation of DNA Replication QUESTIONS 1.What are the requirements for the initiation of DNA replication? 2.How is initiation limited to once per cell cycle? 3.What effect might chromatin structure have on initiation? 4.Overview of the temporal nature of replication initiation Essential for cell growth, development, integrity of the genetic information Each origin initiates replication only once per cell cycle Highly regulated. Commitment to DNA replication is an irreversible decision Many initiation proteins are upregulated in cancer cells

4 DNA Replication Begins at Specific Sequences Fiber Autoradiography Visualize 3 H-Thymidine incorporation following a short pulse

5 origin Isolation of Autonomously Replicating Sequences Restriction digest of yeast chromosomal DNA “Shotgun” clone into a URA3 origin-less plasmid Select for URA+ cells Transform into ura3 yeast

6 Isolation of Autonomously Replicating Sequences EcoRI URA3 URA+

7 Isolation of Autonomously Replicating Sequences EcoRI URA3

8 Isolation of Autonomously Replicating Sequences EcoRI URA3 Ura-

9 Isolation of Autonomously Replicating Sequences EcoRI URA3 -URA plate URA+

10 ARS elements consist of two domains A { B ARS element ~ bp

11 ARS elements share a common sequence - the ACS A { B ARS element ~ bp WTTTAYRTTTW W= A or T R= A or G Y= T or C ARS Core Consensus Sequence

12 Plasmid stability measurements of linker scan mutants A { B ARS element ~ bp 1. Transform plasmid into yeast selecting for URA3 marker Failure to recover high frequency of transformation (HFT) - essential sequences 2. Grow yeast in medium lacking uracil to get a population of cells 3. Grow in medium containing uracil to allow plasmid loss events 4. Calculate percentage of cells containing plasmid after X generations “GGTCGAC” SalI

13 A B1 B2ISIS Linker scan analysis of ARS _ + HFT ACS Percentage of plasmid containing cells

14 ARS1 AB1B2B3 Linker scan analysis reveals modular structure of origins ACS ARS307 Essential Important

15 ARS1 ARS315 ARS305 AB1B2B3 There are at least two broad classes of origins in S. cerevisiae ACS ISIS AB1B2 ARS307 ? Inhibitory element

16 Jacob and Brenner’s Replicon Model WTTTAYRTTTW Initiator protein Replicator Jacob, F., and S. Brenner [On the regulation of DNA synthesis in bacteria: the hypothesis of the replicon.] C R Hebd Seances Acad Sci 256:

17 Jacob and Brenner’s Replicon Model Initiator protein -- DnaA Replicator -- OriC 13mers DnaA boxes E. coli

18 Jacob and Brenner’s Replicon Model Initiator protein -- ORC (Origin Recognition Complex) Replicator -- ARS Bell, SP and Stillman, B. (1992) ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357(6374): AB1B2B3

19 ORC binds to origins of replication origin A B1 B2 ORC -ORC is a six-subunit protein complex that binds to ARS elements. -Specific DNA binding requires ATP -All subunits are essential and are required for initiation -ORC binds ARS elements throughout the cell cycle

20 …and then recruits Cdc6p and Cdt1p during early G1 origin A B1 B2 ORC A B1 B2 ORC Cdc6p Cdt1p

21 Cdc6 is a AAA+ protein required for pre-RC assembly K114A mutant is ts: cdc6-4

22 MCM A B1 B2 ORC pre-RC The “pre-Replicative Complex” assembles at all origins in G1

23 MCM A B1 B2 ORC A B1 B2 ORC Cdc45p Cdc7p-Dbf4ppre-RC Kinases then activate the initiation of DNA synthesis Cdk1-Clb + GINS Ser/Thr protein kinases

24 MCM A B1 B2 ORC Cdc45p Origin unwinding occurs GINS

25 MCM A B1 B2 ORC … followed by the assembly of DNA polymerases Cdc45p Pol  primase GINS and bi-directional DNA synthesis

26 Cdt1 P P Cdk Kinase

27 How do cyclin-dependent kinases prevent pre-RC formation? CyclinB-Cdks phosphorylate ORC, Cdc6p and MCM proteins ORC phosphorylation inhibits its activity through an unknown mechanism Cdc6p phosphorylation causes is proteolysis MCM phosphorylation excludes it from the nucleus, as well as Cdt1p MCM A B1 B2 ORC pre-RC Low Cdk levels A B1 B2 ORC P P P High Cdk levels

28 Cdt1 P P Cdk Kinase

29 ORC and Abf1p position nucleosomes outside ARS1 AB1B2B3 ORC Abf1p

30 … facilitating pre-RC formation at the origin AB1B2B3 ORC Abf1p Cdc6p MCM Cdt1p

31 Origins lacking a B3 element may be sensitive to nucleosome intrusion AB1B2B3 ORC Abf1p AB1B2 ORC Cdc6p MCM Cdt1p

32 AB1B2B3 ORC Abf1p AB1B2 ORC …that inhibits pre-RC assembly Cdc6p MCM Cdt1p Cdc6p Cdt1p

33 ARS1 ARS315 ARS305 AB1B2B3 ACS ISIS AB1B2 ARS307 ? nucleosome I S element may position a nucleosome over origin

34 Chromosome III Position of ARS elements along chromosome III < 10% Inactive

35 Chromosome III Position of ARS elements along chromosome III < 10% Inactive Early origins

36 Chromosome III Position of ARS elements along chromosome III < 10% Inactive Late replicating Telomeres Early origins

37 Replication timing along chromosome VI Raghuraman MK et al. (2001) Replication dynamics of the yeast genome Science 294(5540):

38

39 DNA damage during S-phase, telomeres, late replication and the centromere Questions? Contact Van Andel Research Institute Grand Rapids, MI BMB801 Lecture Dr. Michael Weinreich 10/6/06

40 ARS1 AB1B2B3 Linker scan analysis reveals modular structure of origins ACS ARS307 Essential Important

41 Plasmid stability measurements of linker scan mutants A { B ARS element ~ bp “Linker scans” are typically ~6-10bp in length. They change the existing sequence at multiple base pairs without the adding or deleting nucleotides and introduce a novel restriction site. For example, an 8bp XhoI linker scan across this region might change the existing sequence to CCTCGAGG at each position above.

42 Formation of the pre-RC occurs during G1 origin A B1 B2 ORC A B1 B2 ORC MCM A B1 B2 ORC Cdc6p Cdt1p MCM, DNA helicase ORC, Origin Recognition Complex Cdc6p and Cdt1p, helicase loaders

43 MCM A B1 B2 ORC A B1 B2 ORC Cdc45p Cdc7p-Dbf4ppre-RC Kinases then activate the initiation of DNA synthesis Cdk1-Clb + GINS Ser/Thr protein kinases

44 MCM A B1 B2 ORC … by promoting the assembly of DNA polymerases Cdc45p Pol  primase GINS and bi-directional DNA synthesis

45 QUESTIONS 5. What determines the temporal order of replication during S-phase? 6. How might DNA damage affect DNA replication? 7. What are telomeres and how are they replicated? 8. Overview of the centromere and kinetochore Late replication, the telomere and functions of the centromere

46 MCM A B1 B2 ORC pre-RC The “pre-Replicative Complex” assembles at all origins in G1 Early and late origins assemble the pre-RC during G1 Since some origins are not activated until late S-phase, the regulatory step must occur after pre-RC formation Cdc7-Dbf4 and Cdk1-Clb5,6?

47 Replication timing along chromosome VI Raghuraman MK et al. (2001) Replication dynamics of the yeast genome Science 294(5540):

48 MMS activates the intra-S-phase checkpoint which inhibits late origin firing Mec1p (ATR) Rad53p (Chk2) Chk1p Mec2/Ddc1 (ATRIP) Rad9p DNA damage (MMS) G2/M Inhibit late origin firing WT origin

49 MMS activates the intra-S-phase checkpoint which inhibits late origin firing Mec1p (ATR) Rad53p (Chk2) Chk1p Mec2/Ddc1 (ATRIP) Rad9p DNA damage (MMS) G2/M Inhibit late origin firing WT +MMS origin Slows S-phase

50 Cdc7p-Dbf4p Dbf4p is phosphorylated following replication arrest MCM A B1 B2 ORC Cdc45p Pol  primase GINS DNA damage Replication arrest Mec1p Rad53p Late origins Dbf4p-Cdc7p P ? pre-RC

51 Cdc7p-Dbf4p Homologous proteins in S. pombe, Xenopus, mouse and humans Cdc7p is a serine/threonine kinase required for entry into S phase … after assembly of the pre-RC Dbf4p is a regulatory subunit required for kinase activity Phosphorylates MCM proteins, Cdc45p and polymerase-  primase in vitro Cdc7p-Dbf4p is required for loading Cdc45p and GINS at the origin MCM A B1 B2 ORC A B1 B2 ORC Cdc45p Cdc7p-Dbf4ppre-RC Cdk1-Clb + GINS

52 N M C DBF4 contains three regions of homology among diverse species Is the N-terminus required for viability (DNA replication) or DNA repair? What role might Dbf4p play in checkpoint pathways for DNA repair? Dbf4p

53 The DBF4 N-terminal conserved domain is not required for viability BRDF= BRCT and DBF4 similarity

54 Loss of the BRCT-like domain causes defect in response to DNA damage BRCT M C 1.Promotes DNA repair 2.Is required for firing late replication origins Cdc7p-Dbf4p Dbf4p Essential domain BRCT domains are present in proteins involved in the DNA damage response Protein interaction domains - phospho-peptide binding

55 Mec1p and Rad53p phosphorylate Dbf4p following exposure to HU Mec1p (ATR) Rad53p (Chk2) Chk1p Dun1p Tel1p (ATM) Ddc2 (ATRIP) Cdc5p (Plk) Dbf4p Bub2p Anaphase Crt1 Securin Tem1p (GTPase) Mitotic Exit WT rad hrs in HU Dbf4p Cdc7p DNA repair genes Late origins? DNA damage Slows DNA replication

56 Telomeres occur at the ends of chromosomes 3’ 5’ Nucleosomal Non-nucleosomal TG (1-3) budding yeast T 2 AG 3 human cells Repeated DNA sequence } G-rich

57 The “End Problem” DNA polymerases require a template primer to synthesis DNA No de novo DNA synthesis Polymerases only synthesize DNA in the 5’ to 3’ direction DNA primase can synthesize a short RNA primer without a template, but RNAs are removed during DNA synthesis because they are unstable 5’3’ 5’ RNA Leading Lagging

58 The “End Problem” DNA polymerases require a template primer to synthesis DNA No de novo DNA synthesis Polymerases only synthesize DNA in the 5’ to 3’ direction DNA primase can synthesize a short RNA primer without a template, but RNAs are removed during DNA synthesis because they are unstable 5’3’ 5’ RNA is removed leaving a gap

59 5’3’ If not repaired, chromosomes would shorten over time “Telomere erosion”

60 5’3’ Telomerase is a specialized polymerase that maintains telomere length

61 5’3’ Telomerase is a specialized polymerase that maintains telomere length Telomerase contains an RNA that serves as a template for DNA synthesis Telomerase is an RNA-directed DNA polymerase “Reverse Transcriptase”

62 Telomerase consists of an RNA and a protein component TLC1 is a 1.2kb RNA Est2p is the catalytic subunit of telomerase and belongs to the reverse transcriptase family Telomerase in a Ribonucleoprotein “RNP”

63 Telomerase synthesis at the ends of chromosomes Vega et al. (2003) Nat. Rev. MCB 4:

64 Telomere associated proteins in human cells and budding yeast Blackburn (2001) Cell 106:

65 Telomere loops and folding Vega et al. (2003) Nat. Rev. MCB 4:

66 Conservation of telomerase and associated proteins Vega et al. (2003) Nat. Rev. MCB 4:

67 Telomere associated proteins in human cells and budding yeast Rap1p is a DNA binding protein that recognizes telomeric DNA Rap1p recruits the SIR complex SIR complex recruits Ku Blackburn (2001) Cell 106:

68 The ssDNA-binding protein Cdc13p recruits telomerase to the ends Cdc13p binds ssDNA and also interacts with Est1p Est1p is a subunit of telomerase that binds to Est2 Can bypass requirement for Est1p by making a Cdc13p-Est1p fusion Cdc13p Telomerase Est1p Est2p interaction Blackburn (2001) Cell 106:

69 Ku tethers telomeres to the nuclear periphery in budding yeast In yeast and more complex eukaryotes, telomeres are clustered together and interact with the nuclear periphery In the absence of Ku, telomeres are dispersed throughout the nucleus and no longer show this clustering arrangement

70 Sir2-4 collaborate to form heterochromatin adjacent to telomeres Rap1 Sir3-4 Telomere Rap1 Sir4p Nucleosomal DNA Sir4p also recruits the Sir2p histone deacetylase Rap1p binds to telomeric DNA and recruits Sir3p-Sir4p

71 Sir2-4 collaborate to form heterochromatin adjacent to telomeres Rap1 Sir3-4 Telomere Rap1 Sir4p Nucleosomal DNA Sir2p Sir4p also recruits the Sir2p histone deacetylase Rap1p binds to telomeric DNA and recruits Sir3p-Sir4p

72 Sir2-4 collaborate to form heterochromatin adjacent to telomeres Rap1 Sir3-4 Telomere Rap1 Sir4p Nucleosomal DNA Sir2p Sir4p also recruits the Sir2p histone deacetylase Rap1p binds to telomeric DNA and recruits Sir3p-Sir4p Sir3p-Sir4p bind to ‘hypoacetylated” histones H3 and H4

73 The Sir2p deacetylase is conserved YeastBacterialHuman

74 Sir2-4 collaborate to form heterochromatin adjacent to telomeres Rap1 Sir2-4 Telomere Rap1 Sir4p Sir2-Sir3-Sir4 binding leads to deacetylation of an adjacent nucleosome

75 Sir2-4 collaborate to form heterochromatin adjacent to telomeres Rap1 Sir2-4 Telomere Rap1 Sir4p Sir2-Sir3-Sir4 binding leads to deacetylation of an adjacent nucleosome …. and results in the spreading of the SIR complex along chromatin

76 Sir2-4 Sir2-4 collaborate to form heterochromatin adjacent to telomeres Rap1 Sir2-4 Telomere Rap1 Sir4p Sir2-Sir3-Sir4 binding leads to deacetylation of an adjacent nucleosome …. and results in the spreading of the SIR complex along chromatin

77 Sir2-4 Some ARS elements are within the boundaries of this heterochromatin Rap1 Sir2-4 Telomere Rap1 Sir4p ARS

78 Chromosome III Position of ARS elements along chromosome III < 10% Inactive Late replicating Telomeres Early origins

79 Sir2-4 Rap1 Sir2-4 Telomere Rap1 Sir4p ARS Loss of SIR complex advances replication timing at telomeres Ku Rap1 Telomere Rap1 Sir4p ARS (late) (early) sir3∆ WT Ku

80 Sir2-4 Rap1 Sir2-4 Telomere Rap1 Sir4p ARS Ku (late) Ku70∆ advances replication timing at sub- & telomeric ARSs WT ARS501 (late)

81 Sir2-4 Rap1 Sir2-4 Telomere Rap1 Sir4p ARS Ku Rap1 Telomere Rap1 Sir4p ARS (late) ku70∆ (early) Ku70∆ advances replication timing at sub- & telomeric ARSs Rif1p ARS501 (early) WT ARS501 (late)

82 The centromere and kinetochore CdeI CdeII CdeIII 125bp Centromere

83 The centromere and kinetochore CdeI CdeII CdeIII 125bp Centromere The “Kinetochore” is a proteinaceous structure built upon the centromere for microtubule capture and chromosome segregation ~60 polypeptides

84 The relatively simple centromere of S. cerevisiae is located on an essential 125-bp region of DNA comprising three functional DNA elements. cdeII is AT-rich and can bind to the centromeric nucleosome Cse4/CENP-A; however, the primary determinant of centromere location is cdeIII, which is bound by CBF3 and essential for the localization of all other kinetochore proteins. cdeI is bound by CBF1 and not essential, but its deletion results in chromosome loss. In S. cerevisiae, homologues of three human foundation kinetochore proteins, Mif2/CENP-C, Mtw1/MIS12 and Nnf/CENP-H, exist as part of larger multi- protein complexes – an arrangement that might also apply to higher eukaryotes. Linear models of centromere organization Trends Cell Biol Jul;14(7): Amor DJ, Kalitsis P, Sumer H, Choo KH

85 Three dimensional view of a human centromere/kinetochore Trends Cell Biol Jul;14(7): Amor DJ, Kalitsis P, Sumer H, Choo KH

86 The spindle checkpoint prevents mitosis (anaphase) until all kinetochores attach Mad2p Mad2p-Cdc20p (Inactive) Cdc20p is a key mitotic regulator that is kept inactive until all kinetochores have attached to microtubules MAD1, 2, 3 BUB1, 3 MPS1 Spindle Checkpoint genes

87 Spindle checkpoint prevents anaphase onset by inhibiting cohesin degradation Musacchio and Hardwick Nat Rev MCB (2002)


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