Intro to Chromosome structure

1. Intro to Chromosome structure There’s some odd DNA synthesis that happens at the telomeres because the DNA is linear in eukaryotes. Arm http://www.med.uiuc.edu/m1/genetics/images/webun1/Chromosome.gif

2. The telomere problem users.rcn.com/.../BiologyPages/ T/Telomeres.html With each round of replication, DNA would get shorter.

3. Solution to the telomere problem • An enzyme, telomerase, adds multiple copies of a short sequence to the end of the telomere. It can then be shortened without losing any actual chromosomal DNA, and new copies can be added anytime. • But how can new DNA be added to the end of DNA without a template? Telomerase contains a Guide RNA.

4. Telomerase uses its guide RNA to basepair to the 3’ end, then add bases to that end using its RNA as a template. Telomerase can slide over and repeat this many times, adding many units of DNA. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.figgrp.795

5. Complementary DNA can then be added, lengthening the double stranded end. This DNA synthesis seems to be complicated, involving reverse transcription, slipping of the enzyme, and unusual, weak base pairings. To see a movie go to the bottom of the page of: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.3208

6. G-quartet base-pairing Remaining ss DNA at the end may basepair with itself by an unusual G-quartet pairing, complexed with protein. www.biochemsoctrans.org/. ../bst0290692a01.gif

7. Organization of DNA • Viral nucleic acid (DNA or RNA) • Some circular, some linear • Some double stranded, some single stranded • Very small amount, packed very tightly • Small size is an advantage • Viruses use host cell enzymes, need few genes • Bacterial DNA • Usually single copy of double stranded • Usually circular • Eukaryotic DNA: linear, in several pieces

8. DNA packaging For example, the chromosome of E. coli is 1.2 mm long, but must fit into a bacterium that is only 0.001 mm long! http://www.expatica.com/xpat/xpatsite/www/upload_pix/surprised-face.jpg

9. Protein packaging of DNA • Four proteins in E. coli • Make up 10% of total protein of cell • HU for wrapping; FIS and IHF for bending; HNS for compaction. • Same function as histone proteins in eukaryotes. • Positively charged proteins bind to negatively charged DNA. • End result: nucleoid, a region in the cytoplasm rich in DNA and protein; comparable to a nucleus but without a membrane.

10. DNA of E. coli is supercoiled • In addition to being packaged with proteins, the DNA of E. coli is supercoiled. • Supercoiling. DNA could be “relaxed” or supercoiled. In Eubacteria, DNA is “underwound” (negatively supercoiled); • Supercoiling carried out by topoisomerases. • Example: gyrase, that relieves stress during DNA replication. • Two types, depending on whether 1 or two DNA strands are cut (and repaired) in the process. • http://en.wikipedia.org/wiki/Topoisomerase

11. Supercoiling Top left: relaxed DNA Bottom left: supercoiled. Bottom: schematic of underwinding DNA.

12. Packaging of E. coli DNA Note arrows: one shows where the DNA has been “nicked”, relaxing the supercoiling. The other points to a supercoiled region. That supercoiling can be relaxed in ONE PLACE means that the DNA is constrained in places.

13. The enslaved bacteria • Mitochondria and chloroplasts thought to have originated as prokaryotic endosymbionts in early eukaryotes • Carry out respiratory functions in membrane • DNA is circular, ds DNA like in prokaryotes • Self replicating • Have their own ribosomes, similar to bacterial • Organelle DNA discovered from mutations • Some traits not determined by nuclear genes • Inheritance via mother; ovum has all the cytoplasm

14. Integration of organelles is thorough • Mitochondria: • Replication requires nuclear genes • Polymerases, initiation factors, respiratory proteins are multi-subunit proteins • Several of the subunits for each are nuclear, others are mitochondrial • Chloroplasts • Multi-subunit enzymes jointly encoded • Genes for RuBP carboxylase divided between nucleus and chloroplast http://cellbio.utmb.edu/cellbio/mitoch2.htm

15. Polytene chromosomes Occur in the salivary glands of various flies during development. Condensed areas of DNA line up, produce darkly staining bands. Useful for mapping genes: banding patterns are unique, and in situ hybridization can be used to localize genes on DNA

16. DNA packaging in eukaryotes • Largest human chromosome is made of DNA which is 82 mm long (over 3 inches) • During metaphase, DNA is further compacted to about 10 µm long. • Equivalent to winding 25 miles of spaghetti into a 16 foot canoe. • DNA has to be well packaged to fit into the cell, to be compacted even more during mitosis • still has to be accessible during interphase for use! • Chromatin: grainy appearing mixture of DNA and proteins in the nucelus

17. Nucleosomes: unit of packaging of eukaryotic DNA DNA wrapped around histone proteins: TWO each of the proteins H2A, H2B, H3, and H4. Additionally, H1 on outside helps hold DNA to structure.

18. About histones and arrangement • Histones • positively charged, to attach well to DNA • conserved, very little difference among organisms • How arrangement was determined • DNA collected, treated briefly with nuclease to see how much DNA is protected by proteins • Remove proteins, separate DNA pieces by size on gel • 200 bp pieces of DNA produced • treat more with nuclease, repeat analysis • get 145 bp DNA pieces

19. Structure deduced • the 145 bp of DNA are wrapped around the histone octet which is the core particle. • 200 bp includes region covered by H1 which covers DNA as it enters, exits nucleosome. • the rest of the DNA is linker DNA between.

20. Nucleosomes are wound up Figure shows how “beads on a string” are further wound up to produce a solenoid, the structure of chromatin. During mitosis, this solenoid itself coils further to make chromatids.

21. Organization of DNA • Does DNA packaging create problems? • DNA wrapped tightly around histones • DNA must be accessible for replication, transcription • Modification of histones changes packing with DNA • Acetylation: acetylases added to histones. • Phosphorylation: phosphate groups added by kinases • These groups decrease net positive charges, allow DNA freedom. • Negative supercoiling helps too.

22. Histone acetylation Acetyl group turns + charge into neutral by forming an amide bond. http://web-books.com/MoBio/Free/Ch4G.htm