Plm Cm Biochem Section 1-a

• Chromatin is consist of: • DNA • Structural histone proteins • Non histone proteins • Small quantity of RNA • Within the chromatin the repeating units is the . NUCLEOSOME. 

  is made up of: The for maintaining 146 histones base pairsareofresponsible 2(1.75) suprehelical turns ofthe DNA  chromatins shapea and wrapped around corestructure of 8 histones nucleosomes joined by stretch free The superhelical turnsareprotects theaDNA from of digestion by Adjacent DNA termed "linker DNA" (which varies from 10 - 80 bp a nuclease  in length depending on species and tissue type).

Nucleosome

• Nucleosome is made up of: • • 146 base pairs of 2(1.75) suprehelical turns of DNA wrapped around a core of 8 histones

• • The superhelical turns protects the DNA from digestion by a nuclease

• • The histones are responsible for maintaining the chromatins shape and structure

• • Adjacent nucleosomes are joined by a stretch of free DNA termed "linker DNA"

The nucleosomes contains 4 types of histone proteins: • H2A, H2B, H3, H4 (core histones) • H3, H4 – forms tetramer for the formation of nucleosome • H2A, H2B – forms dimer that stabilizes the primary particle by the the superhelical turns of DNA •  H1 histone appear to stabilize the 30nm fiber, but their position are not clear 



BEADS ON A STRING

M o st o f th e D N A is in re p e a tin g se rie s g ivin g th e so ca lle d “ b e a d s- o n - a - strin g ” a p p e a ra n ce w h e n exa m in e d b y e le ctro n m icro sco p e

• The 10nm fibril is further supercoiled with 6 or 7 nucleosomes per turn to form the 30nm chromatin fibers

30nm chromatin fibers 10nm fibrils

• In order to form mitotic chromosome, the 30nm fiber must be compacted in length in another 100 fold • • The length of each DNA molecule must be compressed about 8000 folds to generate the structure of a condensed metaphase chromosome

Some regions of Chromatin are ACTIVE and others are INACTIVE ACTIVE Nucleosome structure Altered -extensively in DNA Has large regions hypersensitive (100,00 bases long) regions Sensitivity to More sensitive to enzymes digestion by a nuclease (Dnase) Chromatin type EUCHROMATIN (based on transcription ability)

INACTIVE Densely packed No large regions Not affected HETEROCHROMAT IN (constitutive and facultative)

30nm chromatin fibers 10nm fibrils

• DNase I: makes a single strand cuts in any segment of DNA (no sequence speicificity) • • The sensitivity to DNase of chromatin regions being actively transcribed reflects only a potential for transcription rather than transcription itself • • Within the large regions of active chromatin there exist a shorter streches 100-300 nucleotides that exhibit a greater sensitivity to

• In many cases if a gene is capable of being transcribed, it is very often has a Dnase-hypersensitive sites in the chromatin “Immediately upstream”

• • Non histone proteins leads to the formation of hypesensitive sites

• • There are 2 types of heterochromatin • CONSTITUTIVE – always condensed and thus inactive • FACULTATIVE – at times condensed, but other times it is actively transcribed and thus, uncondensed and appears as a euchromatin •

(example is the X-chromosome of females which is active during

• Centromere: is an adenine thymine (A-T) rich region • It binds several proteins with high affinity • This complex is called “KINETOCHORE”, provides the anchor for the mitotic spindle • It thus is an essential structure for chromosomal segregation during mitosis

• The ends of each chromosome contains structures called Telomeres • Human telomeres have a variable number of repeat sequence 5TTAGGG-3 

• TELOMERASE: is the enzyme responsible for telomere synthesis and thus for maintaining the length of the telomere • Telomere shortening has been associated with malignant tumors and aging • This is why telomerase has been an attractive target for cancer chemotherapy and drug

• Telomeres shorten in part because of the end replication problem that is exhibited during DNA replication in eukaryotes only during each replication cycle

• • Because DNA replication does not begin at either end of the DNA strand, but starts in the center, and considering that all DNA polymerases that have been discovered move in the 5' to 3' direction

• • • Telomerase is an enzyme that adds specific DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3' end of DNA strands in the telomere regions

• • • • • The enzyme is a reverse transcriptase that carries its own

• The enzyme telomerase allows for replacement of short bits of DNA known as a telomere, which are otherwise lost when a cell divides via mitosis.

• • In normal circumstances, without the presence of telomerase, if a cell divides recursively, at some point all the progeny will reach their Hayflick limit.

• • The Hayflick limit is the number of times a normal cell population will divide before it stops, presumably because the telomeres reach a critical length.

• • With the presence of telomerase, each dividing cell can replace the lost bit of DNA, and any single cell can then divide unbounded. While this unbounded growth property has excited many researchers, caution is warranted in exploiting this property, as exactly this same unbounded growth is a crucial step in enabling cancerous growth.

• A variety of premature aging syndromes are associated with short telomeres.

• • These include Werner syndrome, Ataxia telangiectasia, Bloom syndrome, Fanconi anemia, Nijmegen breakage syndrome, and ataxia telangiectasia-like disorder.

• • The genes that have been mutated in these diseases all have roles in the repair of DNA damage, and their precise roles in maintaining telomere length are an active area of investigation

• • telomerase therapies may be used not only to combat cancer but also to actually get

Werner syndrome mimics premature aging

• Cancer is a very difficult disease to fight because the immune system has trouble recognizing it, and cancer cells are immortal; they will always continue dividing.

• • Because telomerase is necessary for the immortality of so many cancer types, it is thought to be a potential drug target.

• • If a drug can be used to turn off telomerase in cancer cells, the above process of telomere-shortening will resume—telomere length will be lost as the cells continue to divide,