Cellular Information Flow, The Cell Cycle, And Nucleic Acids

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Cellular information flow, the cell cycle, and nucleic acids

99:163

Medical Biochemistry September 7, 2010

Marc S. Wold 335-6784; [email protected]

Readings: Lippincott (4th ed)- Chapter 29, pp 406-407 Lehninger (5th ed)- Chapter 12, pp 469-479,

Chapter 8

Notes about figures and readings •  Figures shown in Marc Wold’s lectures are from:

–  Nelson & Cox, Lehninger Principles of Biochemistry, 5th ed. 2008

–  Champe et al., Lippincotts Illustrated Reviews: Biochemistry, 4th ed., 2008

Some figures from: –  Alberts et al., Molecular Biology of the Cell, Garland Science,

2003 –  Other sources as indicated

•  Relevant readings from Lehninger and Lippincotts will be noted for each lecture. This material can also be found in any General Biochemistry or Molecular Biology text book.

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Notes about lecture notes •  Each lecture will begin with list of study questions

and key concepts. These concepts are what will be on the exam.

•  Slides have notes that summarize concepts. •  Slides will also have figures to illustrate or

supplement the concepts being presented –  Some figures contain extra information to help give context. These are

labeled as such and will not be on the exam (e.g. for reference only).

•  Color will be used to highlight important concepts.

•  Key vocabulary words will be shown in red and defined.

Outline

•  Overview genetic information pathways

•  Eukaryotic cell cycle –  Cyclin-dependent protein kinases

•  Cellular checkpoints –  Checkpoints and cell cycle regulation

•  Nucleic acids

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1.  What are the stages of the eukaryotic cell cycle? 2.  What is a cyclin-dependent protein kinase? What are the 4

general mechanisms by which cdks are regulated? 3.  What is a cellular checkpoint?

4.  What are the three steps in DNA damage checkpoint activation?

5.  What is the mechanism by which cdks and checkpoint proteins regulate cell entry into S phase? What are the roles of Rb and p53 proteins in this process?

6.  What are the chemical components of DNA and RNA (be able to recognize the bases)?

7.  What is base-pairing? Hoogsteen pairing?

8.  Define the terms in L=T+W. How to the properties defined by these terms relate to each other and affect the structure of DNA?

Study questions & key concepts

Cellular information pathways

•  This section will focus on: –  The organization of

genetic information in the cell

–  How cells copy and utilized genetic information

–  Regulation of information pathways

–  How the environment interacts with information metabolism in the cell

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DNA RNA Proteins

Cellular Information Flow

The Central Dogma proposed that information in cells stored in DNA and then transcribed into RNA from which it was translated into protein.

Replication

DNA Repair

Transcription Translation

RNA processing

DNA RNA Proteins

Protein degradation

Chromatin Structure - organization of DNA in Cell

Cell Cycle regulation - coordination of cellular information flow with other cellular and tissue processes

DNA RNA Proteins


Replication

DNA Repair


RNA processing

DNA metabolism

DNA RNA Proteins

Protein degradation



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The Eukaryotic Cell Cycle •  Eukaryotic cell cycle is composed of four phases

–  G1 (Gap 1) –  S (DNA synthesis) –  G2 (Gap 2) –  M (Mitosis)

–  G0 - Non-dividing cells; terminally differentiated cells - most cells in the body

Figure 12-43. Eukaryotic cell cycle

•  The cell cycle is regulated by cyclin-dependent protein kinases (cdk)

•  Protein kinase - an enzyme that transfers a phosphate group to a protein

•  Cdk activity is needed for progression of the cell cycle

•  Two components: Cdks and cyclins

•  Cdk (grey) is only active when bound to a cyclin molecule (blue). Active site is open in complex.

Cyclin-dependent protein kinase (cdk): the key

regulator of the cell cycle

Figure 12-42. Complex of cdk2 (grey) alone (top) or bound to cyclin (blue-bottom). The active site obstructed by red T loop in absence of cyclin.

ATP (light blue) in active site.

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1.  Cdk is only active when bound to a cyclin molecule –  Different cyclins present

during different parts of the cell cycle.

–  Cyclin synthesis is regulated by cellular growth factors and cytokines

–  Cyclins specifically degraded during the cell cycle (by the ubiquitin pathway).

Regulation of cyclin-dependent kinase

Cells have multiple cyclins •  Different cyclins are present during different parts of the cell

cycle •  All function by interacting with and activating Cdk

•  Mammalian cyclins are named letter designations: cyclin A, B, ...

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•  Signal transduction is conversion of information into a chemical change

•  Signal transduction allows cells to use environmental signals to modify internal processes

•  Pathways usually start with receptors binding molecules at the surface of the cell

•  Signals are amplified by enzyme cascades (often kinases or phosphatases)

Signal transduction

Figure 12-47. Regulation of cell division by growth factors. E2F up regulates synthesis of proteins required for DNA synthesis and some cdks and cyclins.

2.  Cdk and cyclin synthesis regulated cellular growth factors

3.  Cdks are regulated by phosphorylation •  (–) Phosphorylation of a

tyrosine in active site of cdk inactivates kinase

•  (+) Phosphorylation of threonine on T-loop changes conformation and activates kinase

4.  Other regulatory proteins (inhibitors) bind to CDKs and inhibit activity (e.g. p21)

Regulation of cdk (cont.)

Figure 12-47. Regulation of cell division by growth factors. E2F up regulates synthesis of proteins required for DNA synthesis and some cdks and cyclins.

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Example: Cdk regulation of S phase

Figure 12-48. Regulation of the passage from G1 phase to S phase by cdk2.

•  Cdk phosphorylation modulates the activity of proteins essential for each part of the cell cycle:

–  Example 1 - cdks activate spindle formation and changes in morphology of nucleus during mitosis

–  Example 2 (left) - regulation of entry into S phase by cdk2 phosphorylation of Rb (retinoblastoma protein). Active CDK2-cyclinE phosphorylates Rb, reducing its binding to E2F. This activates E2F which up-regulates synthesis of proteins required for S-phase.

Checkpoints •  Cells regulate the cell

cycle in response to internal and external stimuli.

•  Checkpoints prevent cell cycle progression if an essential process is not complete

•  Most cells have checkpoints at: –  the G2-M boundary, –  mitosis, –  in G1 phase and –  in S phase (the intra-S

check point)

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G1 and intra-S Checkpoints •  Types of DNA damage:

base modifications, loss of base, strand breaks, crosslinks

•  G1 checkpoint: DNA damage causes cell cycle to stop at the end of G1

•  Intra-S checkpoint: blocked replication forks or DNA damage cause the cell cycle to stop in S phase

•  Cell cycle resumes after DNA replication is complete or DNA has been repaired

G1/S checkpoint modulates cdk2

Figure 12-48. Regulation of the passage from G1 phase to S phase by cdk2.

Checkpoint activation •  DNA damage activates p53, a

transcription factor that regulates a number of genes including p21.

•  p21 concentration in the cell increases.

•  P21 binds and inhibits cdk2/cyclinE.

•  Rb is not phosphorylated and E2F is not activated so cells to do not enter S phase.

Checkpoint inactivation •  When damage is repaired. p21

levels decline and cdk2/cyclinE becomes active.

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p53 mutations promote proliferation •  Mutations in p53 abolish the

G1 checkpoint! •  Without active p53, cells

with damaged DNA will continue through the cell cycle resulting in an increased mutation rate.

•  More 50% of all human tumors have a mutation in p53.

•  Most p53 mutations arise spontaneously. Individuals that inherit one defective copy of p53 usually have Li-Fraumeni cancer syndrome and develop multiple cancers at a high rate early in life.

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Mechanism of checkpoint signaling Steps in DNA damage checkpoint

pathway: 1.  Sensor proteins (e.g. MRN and

9-1-1 complexes) bind to DNA damage and recruit transducer kinases. Transducer kinases:

1.  ATM (ataxia telangiectasia mutated) 2.  ATR (ATM and Rad3-related)

2.  Transducer kinases amplify signal by phosphorylating effector kinases (ChK1 and ChK2)

3.  Effector kinases phosphorylate protein effectors (p53, cdk2, ….) which modulate cell processes and stop the cell cycle.

Note: ATM & ATR effect cell division and proliferation.

Ashwell, S. et al. Clin Cancer Res 2008;14:4032-4037

Figure for reference only-will not be on test

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Future of Medicine: Prevention of Cancer

•  Chloroquine has been a common anti-malaria drug.

•  Less used currently because of the development of resistant parasites. Maclean et al (2008) Clin. Inv. 118, 79.

“Here we report that intermittent treatment of Myc-overexpressing mice with the antimalarial drug chloroquine (CQ), a compound that activates the ATM kinase and induces p53 without damaging DNA (12, 13), impairs tumor formation and enhances survival.” Michael Kastan’s lab

•  Chloroquine activates ATM kinase in mammalian cells.

•  Active ATM induces p53 and reduces cell division.

Would low level activation of ATM by chloroquine reduce the incidence of cancer in humans?

Future of Medicine: Prevention of Cancer

•  Chloroquine taken regularly appears to reduce the incidence of Burkitt’s lymphoma!

•  Study terminated because minimal effect on malaria.

•  Clinical impact of drugs often unanticipated.

•  How would an cancer preventing drug effect general medical treatment? Figure for reference only

Geser, Brubaker, & Draper (1989) Am J. Epedemiology 129, 740.

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DNA RNA Proteins


Replication

DNA Repair


RNA processing

DNA metabolism

DNA RNA Proteins

Protein degradation



Components of Nucleic Acids

•  Phosphate

•  Sugar (Ribose or 2’ Deoxyribose).

•  Base: Purines and Pyrimidines

•  RNA vs. DNA. –  The chemical difference:

2’H vs. 2’OH –  Differences in structure and

chemistry (e.g. RNA is base labile).

Lodish Figure 2-14

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Nomenclature

RNA DNA Base: adenine Nucleoside: base & sugar adenosine deoxadenosine Nucleotide: base, sugar & phosphate

phosphorylated nucleoside adenylate deoxadenylate monophosphate AMP (or rAMP) dAMP diphosphate ADP (or rADP) dADP triphosphate ATP (or rATP) dATP

Chemical structure of Bases •  Purines in DNA and RNA: Adenine and Guanine •  Pyrimidines in DNA: Cytosine and Thymine •  Pyrimidines in RNA: Cytosine and Uracil •  pKa: 5’-nucleotide

–  A(N1) 3.9 –  C(N3) 4.5 –  G(N1) 10.0 –  U(N3) 10.1 –  dA(N3) 10.5

•  Ionization state varies depending upon pH. Lodish-Figure 4-15

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Polynucleotide Chains have Direction

•  Nucleotide chains are linked by phosphodiester bonds between 5’P and 3’OH.

•  Nucleotide chains have a 5’ end and an 3’ end.

•  Ends are usually terminated by a phosphate or a hydroxide.

Lodish Figure 4-2

Base pairs & the double helix •  Base pairs: G-C, A-T; (RNA A-U) •  Hydrogen bonds: 3 in G-C, 2 in A-T •  Base pairs have similar geometry •  Base pairing causes DNA to form anti-

parallel helix.

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B-form DNA double helix •  Right-handed anti-

parallel helix. 10.4 bp per turn and 3.4Å per bp.

•  Base pairs in center, phosphate-ribose backbone on outside. Base pairs planar.

•  Called Watson-Crick helix (discoverers)

•  Angle between ribose and base leads to formation of major and minor grooves

•  Sequence of bases contains information

Base pair hydrogen bonds •  Hydrogen bonds in double stranded

DNA can be destablized by extremes of temperature and pH

•  Breaking hydrogen bonds in base pairs results in single-stranded (denatured) DNA.

•  This is a reversible reaction. DNA strands that have complementary sequences of bases can anneal to form double-stranded DNA.

•  This property is critical for maintaining genetic information in the cell.

•  This property also allows specific DNA sequences to be identified in the lab (molecular diagnostics).

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UV spectra and denaturation •  The UV absorbance of DNA increases dramatically upon

denaturation. •  The melting temperature for a DNA is its Tm. •  G-C base pairs are more stable than A-T base pairs (#

hydrogen bonds). •  Tm varies primarily with A-T/G-C ratio.

Lodish Figure 4-6

Alternate DNA forms: A, B, and Z • Double stranded DNA is generally in B form. • RNA-DNA duplexes assume A form.

• Z form DNA - a left handed helix. (Rare - forms in alternating purine-pyrimidine sequences; promoted by high salt, specific base modifications (i.e. methylation) and negative supercoiling.)

Table for reference only-will not be on test

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Alternate forms of DNA (and RNA)

•  Complementary inverted sequences (palindomes) can form specialized sequences –  Hairpins –  Cruciforms

•  Other sequences can bend double stranded DNA

Alternate forms of DNA: Hoogsteen pairing

•  Hoogsteen pairing is an additional type of base pair can form with DNA

•  In Hoogsteen pairing T hydrogen bonds with an AT pair or C+ bonds with a GC pair

•  Are most stable at low pH where C is protonated

•  Hoogsteen pairing allows the formation of three stranded and four stranded DNA structures

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Alternate forms: triplex DNA •  Triplex DNA (three stranded DNA or H-DNA) can form

with Hoogsteen pairs and normal base pairs

•  Triplex DNA can form under physiological conditions but only with certain sequences (e.g. all purine in one strand and pyrimidines in the other two strands)

•  Because triplex formation is very specific, sequences can be targeted with triplex forming oligonucleotides

Clinical correlation: Physiology of non-B form DNA

•  Non-B DNA can form in cells but its physiological role is poorly understood.

•  These sequences have been shown to block DNA replication under some conditions and to promote genetic instability

•  A number of genetic diseases are linked to DNA sequences that have the potential for form non-B DNA structures (Z-, H-forms).

–  e.g. Fragile X, Prader-Willi, Mytonic Distrophy, Friedriech’s ataxia and Huntington's

–  90% of patents with Burkitt’s lymphoma have DNA breaks that map to sequences that have the potential to form non-B DNA structures

•  Triplet repeat diseases - Diseases that are caused by expansion of three nucleotide repeats. (Disease state occurs when number of repeats increases above some threshold)

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Coiling of a coil

Compaction of DNA - supercoiling

Controlling compaction of DNA: DNA topology

DNA topology

•  Controlling DNA topology is necessary for cellular DNA metabolism.

•  DNA Topology described by the equation: L = T + W where –  L = linking number –  T = twist –  W = writhe

•  These rules apply to circular DNA and long linear DNA

•  DNA topology affects the behavior of DNA –  Compaction –  Single-strand character –  Formation of non B forms

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DNA Topology-Definitions

Linking Number (L) •  Linking number is defined as

the number of times the two strands are linked together.

•  L is the sum of Watson-Crick twists (Twist) and supercoils (Writhe).

•  L can NOT change without breaking one or both strands of DNA. L is constant (unless something cuts the DNA).

•  DNAs that differ only in a topological property (e.g. L) are called topoisomers

DNA Topology-Definitions Twist Number (T)

•  Twist is the number Watson-Crick turns in a DNA molecule.

•  Twist only depends on number of turns in the double helix

•  For B form DNA, twist is +1 per 10.4 base pairs

•  Most DNA in cells in in B form so generally T = length of DNA/10.4

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DNA Topology-Definitions Writhing Number (W) •  Writhe = supercoiling: coils of

the double helix.

•  Writhe dependent on the path through space

•  Writhe is defined as L-T; the difference between linking number and twist

•  Alternatively Writhe is defined as the number of times the double helix crosses itself in 3 dimensions.

•  DNA with no supercoils (writhe) is called relaxed (far left)

(double helix)

Twist and Writhe interchange freely

•  A change in L can result in either a change in T or W (or both)

•  There is inter-conversion of T and W depending on system conditions

L = T + W

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Underwinding promotes formation of ssDNA & cruciforms

•  Underwound DNA has a lower linking number (L) than relaxed B-form DNA

•  Underwound DNA will have reduced T or reduced W (negative supercoiling) or both

•  The lowest energy state of the helix is B-form so reduced twist results in single stranded regions or formation of cruciforms (for inverted repeat sequences)

•  Cruciform DNA rarely observed in relaxed DNA

Marc S. Wold

99:163 Medical Biochemistry

September 9, 2010

Readings: Lippincott (4th ed)- Chapter 29, pp 395-406

Lehninger (5th ed)- Chapter 24

Higher order DNA structure: chromosome

organization and genome stability

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Outline

•  Chromosome Organization –  The packing problem –  Histones and nucleosomes –  Higher order chromatin structure

•  Genome size and composition •  Genome stability •  Recombination

–  General recombination

–  Site Specific recombination –  Transposons

Study questions & Key concepts

1.  How do non-B forms differ from B-form? 2.  How does genome length compare to cell size?

How is DNA compacted to fit in eukaryotic cells? 3.  What are the properties of histones and what are

their interactions with DNA? 4.  Is a DNA break necessary for homologous

recombination? Why or why not? 5.  What is a Holliday junction? What two classes of

products can be produced when a Holliday junction is resolved?

6.  How are the mechanisms of general recombination, site-specific recombination and transposition similar?

Figure shows E. coli cell and E. coli genome drawn to same scale

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DNA RNA Proteins


Replication

DNA Repair


RNA processing

DNA metabolism

DNA RNA Proteins

Protein degradation



Genomes composed of multiple chromosomes

•  Most genomes are composed of multiple chromosomes

•  Humans 23 pairs of chromosomes (22 homologous pairs and either X & Y or 2 X)

•  Chromosomes must be stably maintained through each cell division (duplicated and segregated)

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The packing problem

•  Genomes are much longer than the cells that contain them. –  Human nucleus (10 µm)

contains human genome (2 m)

•  All organisms compact DNA

•  The information in the compacted DNA must accessible

Figure shows human DNA released from chromosome

Compaction of eukaryotic DNA: nucleosome binding

•  Eukaryotic DNA is organized in nucleoprotein complexes called chromatin

•  Chromatin exists as fibers in which the DNA is wrapped around protein cores called nucleosomes (beads on a string)

•  Nucleosomes composed of histone octamers

•  Chromatin also contains many other proteins

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Histones

•  Histones are small, basic proteins •  They are highly conserved in all eukaryotes •  H2A, H2B, H3 and H4 form the core nucleosome octamer •  There are a number of histone variants that associate with DNA

during certain times of the cell cycle or specific processes •  Histones are subject to many post-translational modifications

(methylation, ADP-ribosylation, phosphorylation, glycosylation and acetylation) which regulate chromatin structure and function (more on this in a later lecture)

Nucleosome core

•  Nucleosome core composed of 2 each of H2A, H2B, H3 and H4

•  One nucleosome every 200 bp •  146 bp wound 1.8x around

core in a solenoid orientation •  H1 binds linker DNA between

cores •  N-terminal tails extend out

from core and are sites of modification/regulation

•  C-terminal tails are short and can also be modified

Nucleosome Structure

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•  Wrapping of DNA around histone core and the action of topoisomerases causes eukaryotic DNA to be underwound.

•  Positioning of nucleosomes is not random. Some sequences bind nucleosomes preferentially.

Topology & chromatin

chromatin structure

•  Binding to nucleosomes compacts DNA 7x

•  Nucleosomes arranged into tightly packed structure called 30 nm fiber.

•  Fibers attached to a non-histone protein “nuclear scaffold”

•  Structure and regularity of 30 nm fiber varies with gene activity, DNA sequence, cell cycle, etc…

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Chromosomal DNA in loops

•  DNA is attached to scaffold to make 20-100 kbp loops

•  Some loops contain related genes

•  Scaffold contains multiple proteins including H1 and topoisomerase II

•  30 nm fiber provides ~100x condensation

Higher order chromatin structure •  Higher order chromatin

structure not well understood but provides more compaction

•  Chromatin structure compacts DNA over 10,000x and also allow access to information content (e.g. transcription) and allow regulation

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Genomes size & genetic information •  There is not a direct correlation between genome size and complexity of

an organism

Table for reference only-will not be on test

Eukaryotic chromosomes

•  Eukaryotic chromosomes are usually very large. Human chromosome range from

•  Centromere = attachment point for proteins that link the chromosome to the mitotic spindle

•  Telomeres = sequences at the end that help to stabilize the chromosome.

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Eukaryotic coding sequences are not contiguous

•  Eukaryotic genes contain both expressed sequences (exons) and non-expressed sequences (introns)

•  In higher eukaryotes intron sequences are often much longer than exon sequences

Sequences in human genome

•  ‘Unique” sequences: –  30% genes (introns & exons) –  (exons 1.5%)

•  Moderate repetitive sequences: –  transposons, –  LINES (long interspersed elements-have ORFs) –  SINES (short interspersed elements-no ORFs) –  retrotransposons, retroviruses

•  Highly repetitive sequences: –  SSR (simple-sequence repeats) –  SD (large-segmental duplications)

•  Other (RNA encoding sequences & other sequences)

(ORF-open reading frame)

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DNA RNA Proteins


Replication

DNA Repair


RNA processing

DNA metabolism

DNA RNA Proteins

Protein degradation



Chromosome stability - Recombination

Genetic recombination •  General classes of recombination:

–  Homologous recombination –  Site specific recombination –  DNA transposition

•  Roles of homologous recombination: –  Meiosis (eukaryotes) –  Double strand break repair (bacteria &

eukaryotic cells) –  Conjugation (bacteria & viruses)

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Recombination in meiosis •  Recombination in meiosis helps stabilize and link sister

chromosomes during meiosis. •  Recombination point can be visualized by light microscopy.

Called crossovers or chiasma.

•  Cancer cells have high rate of mutations and chromosome rearrangements

•  Chromosome painting-hybridization of specific probes each chromosome allows visual identification of each chromosome (multiplex-fluorescence in situ hybridization; M-FISH)

Genetic instability extreme:

cancer cells

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General or Homologous Recombination

•  Homologous recombination is genetic exchange that takes place between homologous DNA sequences.

•  DNA is broken and rejoined to recreate parental or hybrid DNA molecules

•  DNA is exchanged between strands to create heteroduplex molecules.

•  Recombination initiates from either a double-stranded or single-stranded break.

•  Strand invasion leads to the formation of joint molecule - Holliday junction.

Mechanism of homologous

recombination

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•  Strand invasion leads to the formation of joint molecule (Holliday junction).

•  Migration and resolution of Holliday junction leads to exchange of DNA sequences.

•  Catalyzed by recA in bacteria, Rad51 in eukaryotes

•  Resolution of Holliday junction results in: –  Repaired break –  Recombined DNA

Homologous recombination

2 Holliday Junctions

•  Enzymes catalyze branch migration and resolution (by resolvase) of Holliday junctions

•  Movie made by Daniel Brown http://www.youtube.com/watch?v=gQFKdA3VgEg&feature=channel_page

Resolution of Holliday Junctions

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Site-specific recombination

•  Site specific recombination occurs between specific sequences

•  Requires site-specific proteins to catalyze reactions (recombinases & integrases)

•  Cause insertions, deletions and/or inversions

•  Recombinases are enzymes that catalyze recombination between two specific sequences

•  Recombinases bind to DNA (complex shown at right). 4 strands are cut and then rejoined

•  Form Holliday junction-like intermediate

•  Activity related to both topoisomerases and general recombination enzymes

Recombinase

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Antibody diversity is created by “site-specific” recombination

•  Antibody genes are composed of variable, joint and constant segments

•  There are a limited number of genes of each type

•  Each antibody sequence is made by recombination events that combine one V, J and C segment: producing a unique protein

V-J recombination

•  V-J recombination occurs during immune cell maturation (differentiation)

•  Specific recombinases (RAG 1 & 2) catalyze recombination

•  Intervening DNA is lost •  Recombination is

“error-prone”. This results in additional base changes which leads to additional antibody variation.

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Site-specific recombination: viral integration

•  Site-specific recombination is used by viruses to integrate into host genome to create a lysogen

•  Bacteriophage lambda integrates into a specific location on the chromosome (attachment site -attB)

Cre/Lox: gene targeting using site directed recombination

•  Cre recombinase from phage P1 •  It catalyzes the recombination

between loxP sites. •  Used to excise of sequences

between loxP sites. •  Is currently being used to knock

out specific genes in cells and animals

•  There are several other specific recombinase systems in use

•  Figure shows tissue specific knock out of a gene in mice

Science Creative Quarterly, Fall 2004

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Transposons •  Transposons are DNA

sequence that can move in the genome by recombination

•  There are two general classes:

•  DNA transposons –  Mostly found in bacteria –  Recombine into a random site on

the genome

•  Retrotransposons –  Found in most eukaryotes –  Related to retroviruses – have

RNA intermediate –  Some produce virus like particles

(not found outside of cell)

Lodish, Figure10.3

DNA Transposons •  Encode transposase (a

type of recombinase)

•  Recombine into a random site on the genome

•  Often carry antibiotic resistance genes

•  Insertion point can inactivate genes

•  Regulated so that transposition only occurs rarely

Documents

Cellular Information Flow, The Cell Cycle, And Nucleic Acids