As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains Integrity, Even with DNA Breaks.
As a cell pushes or pulls its nucleus through a small constriction, the chromatin must distort and somehow maintain genomic stability despite ever-present double-strand breaks in the DNA. Here we visualize within a living cell the pore-size dependent deformation of a specific locus engineered into chromosome-1 and cleaved. An mCherry-tagged nuclease targets the submicron locus, causing DNA cleavage and recruiting repair factors such as GFP-53BP1 to a large region around the locus. Aspiration of a cell and its nucleus into a micropipette shows that chromatin aligns and stretches parallel to the pore. Extension is largest in small pores, increasing >10-fold but remaining 30-fold shorter than the DNA contour length in the locus. Brochard and de Gennes' blob model for tube geometry fits the data, with a simple modification for chromatin crowding. Continuity of the highly extended, cleaved chromatin is also maintained, consistent with folding and cross bridging of the DNA. Surprisingly, extensional integrity is unaffected by an inhibitor of the DNA repair scaffold.
Physical Sciences Oncology Center at Penn, University of Pennsylvania, Philadelphia, Pennsylvania; Molecular & Cell Biophysics Lab, University of Pennsylvania, Philadelphia, Pennsylvania.
Jerome
J
Irianto
Physical Sciences Oncology Center at Penn, University of Pennsylvania, Philadelphia, Pennsylvania; Molecular & Cell Biophysics Lab, University of Pennsylvania, Philadelphia, Pennsylvania.
Yuntao
Y
Xia
Physical Sciences Oncology Center at Penn, University of Pennsylvania, Philadelphia, Pennsylvania; Molecular & Cell Biophysics Lab, University of Pennsylvania, Philadelphia, Pennsylvania; Graduate Group/Department of Physics & Astronomy, University of Pennsylvania, Philadelphia, Pennsylvania.
Charlotte R
CR
Pfeifer
Physical Sciences Oncology Center at Penn, University of Pennsylvania, Philadelphia, Pennsylvania; Cancer Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
Roger A
RA
Greenberg
Physical Sciences Oncology Center at Penn, University of Pennsylvania, Philadelphia, Pennsylvania; Molecular & Cell Biophysics Lab, University of Pennsylvania, Philadelphia, Pennsylvania; Graduate Group/Department of Physics & Astronomy, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address: discher@seas.upenn.edu.
Dennis E
DE
Discher
D001696:Biomechanical Phenomena; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D042822:Genomic Instability; D055595:Mechanical Phenomena; D016062:Porosity
D002843:Chromatin