Replication, P53 Gene, Histones, Cell Cycle, and Cancer

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Replication, P53 Gene, Histones, Cell Cycle, and
Cancer

• Robert F. Waters, Ph.D.

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Cell Cycle and Histones
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Eukaryotic Nucleus - Structure

• Chromosomes
• Nucleosome
• Contains a nucleosome core particle
• 146 base pairs of supercoiled DNA
• Around a core of eight histone molecules
• Histone core
• Two copies of H2A, H2B, H3 and H4
• Octamer
• H1 resides outside of core
• Linker histone
• Binds to linker DNA
• Connecting one nucleosome core particle to next

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Histones Continued
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Replication
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Conservation of functions and factors that play a
role in replication
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Role of Cdc6 Cdt1 in helicase assembly
Role of other factors ?
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How is the helicase loaded?
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How does the helicase function?
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Restriction to one round of replication per cell
cycle
CDK
Sic1
DDK
Pre-RC assembly
APC
Initiation of DNA replication
CDK
G1
Cdc6 proteolysis
S
M
G2
CDK
Mcm nuclear export
Pre-RC disassembly
Post-replicative state
CDK
Pre-RC reassembly
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Model for Limiting DNA Replication to Once per
Cycle Formation of a funcational replication
origin (green) involves assembly of essential
replication factors (blue spheres) onto DNA at a
site marked by an origin recognition complex
(ORC) (gray spheres). Origin firing (pink)
involves the initiation of replication forks from
assembled origins. Some replication factors
travel with the fork, while others dissociate or
remain at the origin, resulting in a spent
origin. Assembly and firing of origins are
confined to different parts of the cell cycle
(green and pink, respectively), thereby limiting
initiation of DNA replication to once per cell
cycle.
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Rate of DNA synthesis and the need for multiple
origins
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Multiple origins of replication in Eukaryotes

Are all origins created equal?
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Cell cycle control of chromosome duplication
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Early Late firing origins
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Summary

• ORC complex assembles at multiple origins
• ORCs associate with Cdc6 Cdt1 and are
  licensed
• MCM/helicase complex is loaded on to the origin
• Ordered assembly of additional complexes leads to
  a competent pre -Initiation complex
• In S-phase, cell cycle regulated kinases trigger
  replication
• After initiation the pre-IC complex is dismantled
  to prevent premature re-replication without cell
  cycle
• Replication occurs at two classes of origins
  (early late)
• Origins in most eukaryotes are very poorly
  defined
• Novel tools are defining new ARS sites on a
  genome-wide level
• High conservation of function from Bacteria to
  humans -though mechanistic details may differ in
  each species

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DNA Damage Repair
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Replication RNA Synthesis Decoding the Genetic
Code
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• DNA an emblem of the 20th century.
• A simple yet elegant structure a double helix
  with a sugar phosphate backbone linked to 4
  types of nucleotide on the inside that are paired
  according to basic rules. Amazingly this simple
  molecule has the capacity to specify Earths
  incredible biological diversity.
• The double-stranded structure suggests a mode of
  copying (replication) and the long strings of
  the 4 bases encode biological life.
• The human genome is just 3.5 billion base pairs
  and greater than 95 is considered to be
  non-coding (or junk).

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• .
• DNA replication is semi-conservative
  (Meselson-Stahl, 1958).
• Replication requires a DNA polymerase, a
  template, a primer and the 4 nucleotides and
  proceeds in a 5 to 3 direction (Kornberg,
  1957).
• Replication of the Escherichia coli genome (a
  single circular DNA) starts at a specific site
  (ori) and is bi-directional (Cairns, 1963).
• Replication is semi-discontinuous (continuous on
  leading strand and discontinuous on lagging
  strand) and requires RNA primers (Okazakis,
  1968).
• Lagging strand synthesis involves Okazaki
  fragments.
• DNA polymerase III is the replicative enzyme of
  E. coli (Cairns and deLucia, 1969).

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Replication as a Process
1. Double-stranded DNA unwinds.
2. The junction of the unwound molecules is a
replication fork.
3. A new strand is formed by pairing
complementary bases with the old strand.
4. Two molecules are made. Each has one new and
one old DNA strand.
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DNA Replication is Semi-discontinuous
Continuous synthesis
Discontinuous synthesis
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• Primase
• Makes initial nucleotide (RNA primer) to which
  DNA polymerase attaches
• New strand initiated by adding nucleotides to RNA
  primer
• RNA primer later replaced with DNA

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Replication
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
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Replication
DNA polymerase enzyme adds DNA nucleotides to
the RNA primer.
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Replication
DNA polymerase enzyme adds DNA nucleotides to
the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
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Replication
Leading strand synthesis continues in a 5 to 3
direction.
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Replication
Leading strand synthesis continues in a 5 to 3
direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.
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Replication
Overall direction of replication
3
5
3
5
Okazaki fragment
3
5
5
3
3
5
Leading strand synthesis continues in a 5 to 3
direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.
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Replication
3
5
3
5
3
5
3
5
3
3
5
5
Leading strand synthesis continues in a 5 to 3
direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.
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Replication
3
3
5
Leading strand synthesis continues in a 5 to 3
direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.
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Replication
Exonuclease activity of DNA polymerase I removes
RNA primers.
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Replication
Polymerase activity of DNA polymerase I fills the
gaps. Ligase forms bonds between sugar-phosphate
backbone.
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DNA Synthesis
Synthesis on leading and lagging strands
Proofreading and error correction during DNA
replication
Simultaneous replication occurs via looping of
lagging strand
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Simultaneous Replication Occurs via Looping of
the Lagging Strand
Helicase unwinds helix SSBPs prevent
closure DNA gyrase reduces tension Association
of core polymerase with template DNA
synthesis Not shown pol I, ligase
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Procaryotic (Bacterial) Chromosome Replication
Bidirectional Replication Produces a Theta
Intermediate
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P53 Gene
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P53 Gene

• THE p53 GENE is a tumor suppressor gene, i.e.,
  its activity stops the formation of tumors. If a
  person inherits only one functional copy of the
  p53 gene from their parents, they are predisposed
  to cancer and usually develop several independent
  tumors in a variety of tissues in early
  adulthood. This condition is rare, and is known
  as Li-Fraumeni syndrome. However, mutations in
  p53 are found in most tumor types, and so
  contribute to the complex network of molecular
  events leading to tumor formation.   

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P53 Continued

•  The p53 gene has been mapped to chromosome 17.
  In the cell, p53 protein binds DNA, which in turn
  stimulates another gene to produce a protein
  called p21 that interacts with a cell
  division-stimulating protein (cdk2). When p21 is
  complexed with cdk2 the cell cannot pass through
  to the next stage of cell division. Mutant p53
  can no longer bind DNA in an effective way, and
  as a consequence the p21 protein is not made
  available to act as the 'stop signal' for cell
  division. Thus cells divide uncontrollably, and
  form tumors.

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Other Tumor Suppressors

• THE PANCREAS is responsible for producing the
  hormone insulin, along with other substances. It
  also plays a key role in the digestion of
  protein. There were an estimated 27,000 new cases
  of pancreatic cancer in the US in 1997, with
  28,100 deaths from the disease.
• About 90 of human pancreatic carcinomas show a
  loss of part of chromosome 18. In 1996, a
  possible tumor suppressor gene, DPC4 (Smad4), was
  discovered from the section that is lost in
  pancreatic cancer, so may play a role in
  pancreatic cancer. There is a whole family of
  Smad proteins in vertebrates, all involved in
  signal transduction of transforming growth
  factor-beta (TGF-beta) related pathways.

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Hereditary Prostate Cancer

• THE SECOND LEADING cause of cancer death in
  American men, prostate cancer will be diagnosed
  in an estimated 184,500 American men in 1998 and
  will claim the lives of an estimated 39,200.
  Prostate cancer mortality rates are more than two
  times higher for African-American men than white
  men. The incidence of prostate cancer increases
  with age more than 75 of all prostate cancers
  are diagnosed in men over age 65.
• Despite the high prevalence of prostate cancer,
  little is known about the genetic predisposition
  of some men to the disease. Numerous studies
  point to a family history being a major risk
  factor, which may be responsible for an estimated
  5-10 of all prostate cancers.   

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Ras Oncogene

• Cancer occurs when the growth and differentiation
  of cells in a body tissue become uncontrolled and
  deranged. While no two cancers are genetically
  identical (even in the same tissue type), there
  are relatively few ways in which normal cell
  growth can go wrong. One of these is to make a
  gene that stimulates cell growth hyperactive
  this altered gene is known as an 'oncogene'.
• Ras is one such oncogene product that is found on
  chromosome 11. It is found in normal cells, where
  it helps to relay signals by acting as a switch.
  When receptors on the cell surface are stimulated
  (by a hormone, for example), Ras is switched on
  and transduces signals that tell the cell to
  grow. If the cell-surface receptor is not
  stimulated, Ras is not activated and so the
  pathway that results in cell growth is not
  initiated. In about 30 of human cancers, Ras is
  mutated so that it is permanently switched on,
  telling the cell to grow regardless of whether
  receptors on the cell surface are activated or
  not.
• Usually, a single oncogene is not enough to turn
  a normal cell into a cancer cell, and many
  mutations in a number of different genes may be
  required to make a cell cancerous. To help
  unravel the intricate network of events that lead
  to cancer, mice are being used to model the human
  disease, which will further our understanding and
  help to identify possible targets for new drugs
  and therapies.

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Colorectal Cancer

• A TP53 polymorphism is associated with increased
  risk of colorectal cancer and with reduced levels
  of TP53 mRNA Federica Gemignani1,2,5, Victor
  Moreno3,5, Stefano Landi1,2,5, Norman Moullan1,
  Amélie Chabrier1, Sara Gutiérrez-Enríquez1, Janet
  Hall1, Elisabeth Guino3, Miguel Angel Peinado4,
  Gabriel Capella3 and Federico Canzian1
• We undertook a case-control study to examine the
  possible associations of the TP53 variants
  ArggtPro at codon 72 and p53PIN3, a 16 bp
  insertion/duplication in intron 3, with the risk
  of colorectal cancer (CRC). The p53PIN3 A2 allele
  (16 bp duplication) was associated with an
  increased risk (OR 1.55, 95 CI 1.10-2.18,
  P0.012), of the same order of magnitude as that
  observed in previous studies for other types of
  cancer. The Pro72 allele was weakly associated
  with CRC (OR1.34, 95 CI 0.98-1.84, P0.066).
  The possible functional role of p53PIN3 was
  investigated by examining the TP53 mRNA
  transcripts in 15 lymphoblastoid cell lines with
  different genotypes. The possibility that the
  insertion/deletion could lead to alternatively
  spliced mRNAs was excluded. However, we found
  reduced levels of TP53 mRNA associated with the
  A2 allele. In conclusion, the epidemiological
  study suggests a role for p53PIN3 in
  tumorigenesis, supported by the in vitro
  characterization of this variant.

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Gene Therapy

• p53 tumor suppressor gene therapy for
  cancer.Roth JA, Swisher SG, Meyn
  RE.Department of Thoracic and Cardiovascular
  Surgery, University of Texas, M. D. Anderson
  Cancer Center, Houston, USA.Gene therapy has
  the potential to provide cancer treatments based
  on novel mechanisms of action with potentially
  low toxicities. This therapy may provide more
  effective control of locoregional recurrence in
  diseases like non-small-cell lung cancer (NSCLC)
  as well as systemic control of micrometastases.
  Despite current limitations, retroviral and
  adenoviral vectors can, in certain circumstances,
  provide an effective means of delivering
  therapeutic genes to tumor cells. Although
  multiple genes are involved in carcinogenesis,
  mutations of the p53 gene are the most frequent
  abnormality identified in human tumors.
  Preclinical studies both in vitro and in vivo
  have shown that restoring p53 function can induce
  apoptosis in cancer cells. High levels of p53
  expression and DNA-damaging agents like cisplatin
  (Platinol) and ionizing radiation work
  synergistically to induce apoptosis in cancer
  cells. Phase I clinical trials now show that p53
  gene replacement therapy using both retroviral
  and adenoviral vectors is feasible and safe. In
  addition, p53 gene replacement therapy induces
  tumor regression in patients with advanced NSCLC
  and in those with recurrent head and neck cancer.
  This article describes various gene therapy
  strategies under investigation, reviews
  preclinical data that provide a rationale for the
  gene replacement approach, and discusses the
  clinical trial data available to date.

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Example of Pattern Recognition

• Cancer

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Idealized expression pattern
Neighborhood analysis
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Class Predictor

• The General approach
• Choosing a set of informative genes based on
  their correlation with the class distinction
• Each informative gene casts a weighted vote for
  one of the classes
• Summing up the votes to determine the winning
  class and the prediction strength

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Validation of Gene Voting
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Proteonomics
PROTEIN MICR
Templin et al. 2002 Trend in Biotch. Vol 20