Lecture 10 Chapter 9 Chromosomes Chromosomes together o Function of centromere is important holds two sister chromatids Hold two chromosomes together during crossing over as well Also important for maintaining genomic information o Two copies of the same chromosome homologous chromosome Centromeres poles o Sister chromatid adhesion separated by their centromere to opposite o Pairing homologous chromosomes o Separation of chromosomes during mitosis and meiosis Microtubules pull the centromeres apart at mitosis 2 4 13 Telomere p arm pettie Centromere q arm Telomere Sis te r ch ro Hom m olog ati ous ds chro mos ome s Centromeres with attached chromosome are pulled apart toward pole during mitosis Centromere is region of chrom responsible for segregation during mitosis meiosis Mitrotubues o Responsible for connecting at centromere pulling the chromosomes apart during mitosis o 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 function o 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 Kinetochore o 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 together Microtubules pull the centromeres apart at mitosis o Microtubule Organizing Center MTOC Organizes the microtubules and pulls them with force Located at end of cell Pulls mirotubules pulling chromosomes apart Sister chromatids Microtubules Outer kinetochore Inner Centromeric binding proteins Micro tubul e organ izing cente kinetochore r S cerevisiae Centromeres MTO Is a very short and specific sequence o Centormere is a short sequence CEN fragment C A T rich Sufficient to allow segregation of plasmid Every chromosome has CEN region o Centromeres serve only to attach chromosome to spindle fiber Plays only role in distinguishing one chromosome from another o Point mutations reduce centromere funciton o Three Types of Sequence Elements 1 CDE 1 2 CDE 2 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 found in all centromeres function depends on length rather than exact sequence 90 is A T rich 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 binds CDE 1 part of sequence binds CDE 3 part of sequence binds CBF1 and CBF3 proteins to sequence and CBF1 CBF3 CtF19 microtubules complex structure Mcm21 Cse4 forms complex all in one unit special histone binds to DNA and organizes it into binds CDE2 region to Cse4 CenH3 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 Centromeres Nuclease resistant Binds CDE 1 Binds CBF1 CBF3 Microtubules Binds CDE III Cse4 Special histone CenH3 Binds CDE II o central domain is the main component of the centromere o have to form compact DNA structure centromere so chromatids can withstand the pulling by the Outer repeats Central domain Heterochromatin microtubules Human Centromere Telomeres o Composed of alpha satellite long repeats o One sequence 171 bp and are repeated 100 times o Very critical for maintaining the centromere structure o Are located at the ends of most eukaryotic linear chromosomes Maintains integrity of linear structure Prokaryotic chromosomes are circular so don t have telomeres o 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 sequence o 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 quartets o Asymmetric sequence G rich strand o Capable of regenerating intra strand interactions Folding on it self to form G quartets o This stabilizes the single stranded DNA Telomere Looping o 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 strands and pairs with it this occurs in DNA repair telomeres and sometimes circular DNA o When this occurs with the telomeres creates the T loop where the end of the main T loop 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 damage o 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 G T Rich 5 5 3 C A Rich 3 D loop 5 5 3 3 Telomere