2/4/13Lecture 10Chapter 9 - Chromosomes• Chromosomeso Function of centromere is important  holds two sister chromatids together Hold two chromosomes together during crossing over as well Also important for maintaining genomic informationo Two copies of the same chromosome = homologous chromosome• Centromereso Sister chromatid adhesion  separated by their centromere to opposite poles o Pairing homologous chromosomes o Separation of chromosomes during mitosis and meiosis  Centromeres with attached chromosome are pulled apart toward pole during mitosis  Centromere is region of chrom. responsible for segregation during mitosis & meiosis• Mitrotubueso Responsible for connecting at centromere & pulling the chromosomes apart during mitosiso Microtubules are composed of a cellular filamentous system  It is reorganized during mitosis into spindle which pulls the chromosomes to poles o Microtubules connect to centromere by two things: Microtubule binding proteins  Centromeric binding proteins • These two groups hold centromere and microtubules together• Critical for functiono Centromere is identified by a DNA sequence that binds specific proteins Don’t bind to microtubules but establish site and which the mitrotubule binding proteins attach and bind the centromere and microtubule together • Kinetochoreo Centromeric DNA binds specific proteins that are responsible for establishing the structure that attaches chromosome to microtubues  kinetochore Holds together centromere and microtubules o Contains two parts: 1. Outer kinetochore  connects spindle fiber 2. Inner kinetochore  connects centromere o it is the centromeric binding proteins that hold the two sister chromatids togethero Microtubule Organizing Center (MTOC) Organizes the microtubules and pulls them with force Located at end of cell Pulls mirotubules, pulling chromosomes apart• S. cerevisiae Centromereso Centormere is a short sequence  CEN fragment Is a very short and specific sequence A-T rich Sufficient to allow segregation of plasmid Every chromosome has CEN regiono Centromeres serve only to attach chromosome to spindle fiber Plays only role in distinguishing one chromosome from anothero Point mutations reduce centromere funcitono Three Types of Sequence Elements 1. CDE 1: • cell cycle dependent element• located at left boundary (left side of sequence)• a pint mutation only reduces efficiency of centromere, does not cause it to lose its function 2. CDE 2: • >90% is A-T rich• found in all centromeres• function depends on length rather than exact sequence • Located in middle of sequence • Point mutation only reduces efficiency of centromere, does not cause it to lose its function 3. CDE 3: • highly conserved• located at right boundary of all centromere (end of sequence)• a point mutation in the region causes a complete loss of function***critical for centromere function***o Centromere Binding Proteins in S. cerevisiae (Yeast) CBF1 binds CDE-1 part of sequence CBF3 binds CDE-3 part of sequence CtF19 binds CBF1 and CBF3 proteins to sequence and microtubules Mcm21 forms complex; all in one unit Cse4 special histone  binds to DNA and organizes it into complex structure • CenH3 binds CDE2 region to Cse4• Organizes the kinetochore• Outside kinetorchore, the spindle attaches Proteins bound to CDE1, 2, 3 interact with another group of proteins  ctf19• links the centromeric complex to kinetochore proteins and microtubules Binding of DNA at this structure allows proteins bound to this structure to become part of a single complex • Scaffold for assembly of kinetochore linking centromere and microtubule• S. pombe Centromereso central domain is the main component of the centromereo have to form compact DNA structure (centromere) so chromatids can withstand the pulling by the microtubules• Human Centromereo Composed of alpha-satellite (long repeats)o One sequence = 171 bp and are repeated ~100 timeso Very critical for maintaining the centromere structure• Telomeres o Are located at the ends of most eukaryotic linear chromosomes Maintains integrity of linear structure  Prokaryotic chromosomes are circular, so don’t have telomereso Function: to protect and stabilize the ends of the chromosomes o If remove telomeres, chromosome becomes unstable and produces sticky ends This would allow two chromosomes to be able to stick together o Telomeres are composed of simple repeats of DNA and are the same on all chromosomes o DNA FISH can label chromosomes using telomere specific sequenceo Consists of long series of short, tandemly repeated sequences – 100-1000 repeats depending on organism One strand consists of C-A rich Other strand consists of G-T rich*asymmetric sequence*o The C-A strand is more prone for degradation which leaves a overhanging of DNA that is G-T rich So telomere have a G-T overhang Length of 3’ overhang varies  ~100-200nt in humans• G-quartetso Asymmetric sequence  G rich strando Capable of regenerating intra-strand interactions  Folding on it self to form G-quartetso This stabilizes the single stranded DNA • Telomere Loopingo D-loop: (displacement loop) occurs when the two strands of the dsDNA are separated for a certain stretch of sequences and held apart by a 3rd stretch of DNA  the 3rd stand has a base sequence that is complementary to one fo the main strands and pairs with it  this occurs in DNA repair, telomeres, and sometimes circular DNAo When this occurs with the telomeres, creates the T-loop, where the end of the chromosome is joined to a sequence at an earlier point on the chromosome The 3’ inner DNA strand invades the earlier 3’ outer loop causes the inner strand at that same invasion point to create a D-loop. • A special protein stabilizes this point The T-loop, which is completed by the D-loop, protects the ends of the chromosome form damageo Requires TRF2  telomere repeat binding factor 2 Loop formation catalyzed by TRF2 Together with other proteins, forms a complex that stabilizes chromosome ends• Protects ends bc deletion of TRF2 causes chromosome rearrangements to occur• Telomere Shorteningo Bc lagging strand is synthesized by creation of okasaki fragments, when DNA replication reaches the end of the strand, there isn’t enough space to create a primer for