DNA Replication II

Lecture .4By

Harmand A. HamaMSc. In Molecular Biology

Objectives:

• DNA Replication-Elongation and Termination.

• To figure out what the lagging and leading strand are?

• To understand the telomers and telomerase.

DNA ReplicationMechanism

v Initiation

v Elongation

v Termination

DNA Replication:Elongation• DNApolymeraseIII (DNAPIII) catalyzesthe elongation of DNA molecules

by adding nucleotides to the 3’ end of a pre-existing nucleotide• As each nucleotide is added, the last two phosphategroupsare

hydrolyzed to form pyrophosphate.Ø Pyrophosphateisbroken down into two phosphatesØ NTP NMP +2P

DNA Replication:Elongation

Fig. 16.11

DNA Elongation

DNAPolymerase

DNA polymerase I (DNAP I) replaces the RNA primer with DNAcomplementary to the template

DNAElongation

DNAP III:elongates DNAstrand

DNAP I:replaces RNA with DNA

Fig. 16.14

DNA polymerase III

DNA polymerase I

The problem at the fork

• Due to antiparallel nature ofDNA

• One parental strandhas its 3’ end at the fork while the otherparental strand has its 5’ end at the fork.

• But DNA synthesis can onlyproceed ina5’-3’ direction.

ReplicationFork

Fig. 16.16http://kvhs.nbed.nb.ca/gallant/biology/replication_overview.jpg

Directionality

• A strand of DNA can only addnucleotidesonto its 3’ end

• DNA elongation only proceedsin the 5’to 3’ direction

• DNAP must move along thetemplate strand’s 3’to 5’direction.

The leading and laggingstrands

• Leading strand: is synthesized continuously• Lagging strand: is synthesized in short, discontinuous

segments of1000-2000 nucleotides called Okazakifragments

Lagging strand: Okazakifragments

DNAP III synthesizes the DNADNAP I replaces the RNA primer

withDNA complementary tothe template

DNA ligase joins broken pieces of DNA by catalyzing the formation of phosphodiesterbonds

DNA Ligase

http://fhs-bio-wiki.pbworks.com/f/DNA%20Ligase%20reaction.jpg

DNA ReplicationMachinery

III

II

III

Fig. 16.15

DNA Replication:Elongation

DNA replication:Termination

DNA replication endswhen:Ø Reachthe end of the chromosomeØ Replication bubble / fork meets

another replicationbubble/ fork

Final Step - Assembly into Nucleosomes

• As DNA unwinds, nucleosomes must disassemble

• Histones and the associated chromatin proteins must be duplicated by new protein synthesis

• Newly replicated DNA is assembled into nucleosomes almost immediately.

• Histone chaperone proteins control the assembly

Telomeresü Telomeres are made of repetitive sequences of non-coding DNA that

protect the chromosome from damage. Each time a cell divides, thetelomeres become shorter. Eventually, the telomeres become soshort that the cell can no longer divide.

ü Problemat the endsü Agingü Telomerase

http://images.sciencedaily.com/2009/10/091005110401-large.jpg

The problem with replicationat the ends of linearDNA

Ø DNA gets progressivelyshorter with each round ofreplication

Ø Prokaryotes avoid problembyhaving circular DNA

Telomere

Structure:• DNA found at the ends of eukaryotic

chromosomes• Noncoding (no genes)• Consists of multiple repeats of

a short genetic sequence (humans: TTAGGG)

http://www.dreva.com/shop/images/telomeres.jpg

Why telomeres are excited?!

� Function:Ø Protect chromosomes from being eroded through multiple

rounds of DNAreplicationØ Less about preserving genetic information and more about

serving asa protective cap to prevent unwinding because� uncapping is sensed bycells� leads to cellular aging where cells stop growing and

dividing (senescence) and/or programmed self-destruction (apoptosis)

Telomere andAging

Weng, Nan-ping. "Telomere and adaptive immunity." Mechanisms ofAgeing andDevelopment129.1-2 (2008): 60-66. Fig. 1.Model of telomere attrition in Tand Bcells with age.Lossof telomere length is rapid during the first decade of lifeand decreases during most of adult life. At advanced age, the rate of telomere shortening may increase. The graph projects the telomere attrition in CD4,CD8, and B cells in vivo based on the cross-sectional analysis of telomere length in lymphocytes with age. Whether significantly shortened telomeres in advanced age cause declined function of lymphocytes will need further study.

http://www.sierrasci.com/telomere/index.html The time remaining on this "telomere clock" can be measured from our blood cells. When such measurements are taken, a significant correlation is found between a person's age and the number of "ticks" remaining on the person's clock.

Telomerase

• Enzymeresponsiblefor addingtelomeres to chromosomes

• Aribonucleoproteinthat extendstheendsof chromosomes using the enzymatic action of reversetranscriptase

http://www.ibioseminars.org/lectures/cell-bio-a-med/elizabeth-blackburn.htmlLecture 1 –Telomeres &Telomerase (48:27)Lecture 2 –Telomeres & Telomerase in Human Stem Cells &Cancer (26:58)Lecture 3–Stress,Telomeres & Telomerase in Humans (45:58)

Telomerase andAging

• Telomerase is only found in certain cellsØ Germ line cellsØ Cancercells

• Cells that have telomerase live for a longerperiod of time

• Thelackof telomerase in most cellsmay explain why cellshaveafinite lifespanØ Example:DNAof dividing somatic cells (non-sexcells)

tend to beshorter in older individuals

Videos: Telomere andAging

� http://www.youtube.com/watch?v=xI70O69EZY8 (Today Show "How to live to 100", 5:59)

� http://www.youtube.com/watch?v=m3qqUy880dQ(Isagenix, 11:08)

� http://www.youtube.com/watch?v=lBngws_cWho (TedMed, 12:42)

� http://www.youtube.com/watch?v=-bmMv6dcsgE(Independent Pharmacy Business GrowthConference, February 23, 2012 in Orlando, FL, 1:44:06)

References

• Cox, M. M., Doudna, J. A., & O'Donnell, M. (2012). Molecular biology: principles and practice (p. 809). New York, NY. USA:: WH Freeman and Company.

• Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Scott, M. P., Bretscher, A., ... & Matsudaira, P. (2008). Molecular cell biology. Macmillan.

• Mather, K. A., Jorm, A. F., Parslow, R. A., & Christensen, H. (2011). Is telomere length a biomarker of aging? A review. Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences, 66(2), 202-213.

• Opresko, P. L., & Shay, J. W. (2017). Telomere-associated aging disorders. Ageing research reviews, 33, 52-66.